Devices and methods for analyzing biological samples

ABSTRACT

Described herein are systems and methods for analyzing biological samples. Including a method for processing an analyte, comprising providing a fluidic device comprising the analyte and one or more polymer precursors; selecting a discrete area within said fluidic device; providing an energy source in optical communication with fluidic device; and selectively supplying a unit of energy generated from the energy source to the fluidic device to generate a polymer matrix within the fluidic device, wherein the polymer matrix is within the discrete area or adjacent to the discrete area.

CROSS REFERENCE

This application is a continuation of International Application No.PCT/US2022/11720, filed Jan. 7, 2022, which claims priority from U.S.Provisional Application No. 63/135,463, filed Jan. 8, 2021; and U.S.Provisional Application No. 63/253,500, filed Oct. 7, 2021. Each of theforegoing applications is incorporated herein by reference in itsentirety.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BACKGROUND

In the field of cellular biology, single-cell analysis can include thestudy of genomics, transcriptomics, proteomics, metabolomics, andcell-cell interactions at the single-cell level. Due to theheterogeneity seen in both eukaryotic and prokaryotic cell populations,analyzing a single cell can make it possible to discover mechanisms notseen when studying a bulk population of cells. In single-cell analysis,changes in single cells can be tracked or observed at the level ofgenes, proteins, or other cellular components. For example, in cancer,where cells may be mutating, it can be of interest to see how cancerschange at the genetic level. These patterns of somatic mutations andcopy number aberrations can be observed using single-cell sequencing.

SUMMARY

Recognized herein is a need for compartmentalizing components of abiological sample to perform one or more assays on an individualcomponent within a compartment. The one or more assays may be performedwith or without the need for additional processing of the individualcomponent (e.g., without the need for nucleotide amplification steps),while retaining spatial information of the individual components.Compartments may be generated or deconstructed on demand to localize(that is, to constrain to a compartment) or release targeted componentsof the biological sample.

Provided herein is a method comprising: providing a fluidic devicecomprising an analyte and one or more polymer precursors; identifying adiscrete area within the fluidic device; and selectively supplying aunit of energy generated from the energy source to the fluidic device togenerate a polymer matrix from said one or more polymer precursorswithin the fluidic device, wherein the polymer matrix is within thediscrete area or adjacent to the discrete area. In some embodiments, thefluidic device comprises a flow channel. In some embodiments, thefluidic device comprises an open configuration, for example, as shown bythe embodiment of FIG. 20. In some embodiments, the fluidic devicecontains one or more discrete locations wherein the one or more discretelocations are not in fluidic communication with another discretelocation. In some embodiments, the one or more discrete locations areone or more wells on a surface of plate. In some embodiments, whendiscrete locations are defined by wells or cavities or containers in oron a surface, the one or more discrete locations are open at the top.That is, in some embodiments, the one or more discrete locationscomprise an analyte or biological component. In some embodiments, a unitof energy is an amount of energy, such as light energy, effective tocause the synthesis of chambers in a channel. A value of a unit ofenergy for a given embodiment may vary widely depending on, but notlimited to, such factors as the nature of the spatial energy modulatingelement, the size of a channel, the size and geometry of the desiredchambers, the nature of the polymer precursors, and the like.

In some embodiments, the fluidic device further comprises a spatialenergy modulating element. In some embodiments, the fluidic devicecomprises a surface having a capture probe immobilized thereto, whereinthe capture probe couples to the analyte to immobilize the analyte tothe surface. In some embodiments, the polymer matrix is generatedadjacent to or surrounding the analyte to immobilize the analyte. Insome embodiments, the polymer matrix forms a hydrogel. In someembodiments, the capture probe comprises one or more functional groupscapable of interacting with the analyte. In some embodiments, the one ormore functional groups comprise a complimentary DNA sequence to targetDNA or RNA.

In some embodiments, the energy source is in optical communication withsaid fluidic device. In some embodiments, the spatial energy modulatingelement is a light generating device, such as a digital micromirrordevice. In some embodiments, the light generating device generates lightat 350 nm to 800 nm. In some embodiments, the light generating devicegenerates light at 350 nm to 600 nm. In some embodiments, the lightgenerating device generates light at 350 nm to 450 nm. In someembodiments, the light generating device generates UV light. In someembodiments, selectively supplying a unit of energy generated from theenergy source to the fluidic device to generate a polymer matrix withinthe fluidic device is performed using a spatial energy modulatingelement that is a spatial light modulator (SLM). In some embodiments,the SLM is a digital micromirror device (DMD). In some embodiments, theSLM is a laser beam steered using a galvanometer. In some embodiments,the SLM is liquid-crystal based. Light energy from a spatial lightmodulator or spatial energy modulating element may be used tophoto-crosslink polymer precursors to form polymer matrix walls thatmake up chambers formed in a channel.

In some embodiments, the area of the discrete area is less than the areaof the fluidic device. In some embodiments, the analyte is capturedwithin the discrete area. In some embodiments, the size and shape of thediscrete area is adjustable according to the analyte size, the analyteshape, or other properties of the analyte. In some embodiments, analgorithm is used to determine the shape and size of the discrete area.In some embodiments, the algorithm is a supervised, a self-supervised,or an unsupervised learning algorithm. In some embodiments, the discretearea is identified optically. In some embodiments, the discrete area isidentified using a detector configured to detect the location of theanalyte within from an image from light collected from the fluidicdevice. In some embodiments, the detector configured to detect thelocation of the analyte within the fluidic device is a microscopeobjective for imaging the fluidic device. In some embodiments, analgorithm is used to determine where the analyte is located based on theimaging. In some embodiments, the imaging is bright-field imaging,phase-contrast imaging, or fluorescence imaging, or any combinationthereof. In some embodiments, the algorithm is a supervised, aself-supervised, or an unsupervised learning algorithm. In someembodiments, the objective is coupled to an energy source to emit energyto the discrete area in the fluidic device.

In some embodiments, the method further comprises introducing one ormore reagents to the polymer matrix that react with the analyte. In someembodiments, the one or more reagents flow through a membrane. In someembodiments, the membrane is semi-permeable. In some embodiments, themembrane comprises pores. In some embodiments, the pores are less than10 um. In some embodiments, the one or more reagents comprise one ormore of the following: an enzyme, a drug molecule, oligonucleotide,primer, or any combination thereof. In some embodiments, the one or morereagents is a lysis reagent, for example, for lysing cells that areselectively to be removed from the channel of the fluidic device. Insome embodiments, the one or more reagents is a nucleic aciddenaturation reagent. In some embodiments, the one or more reagentsdegrades the polymer matrix.

In some embodiments, the analyte is a cell component. In someembodiments, the analyte is a small molecule composed of nucleic acids,amino acids, intracellular proteins, surface proteins, secretedproteins, exosomes, metabolites or lipids, or any combination thereof.In some embodiments, the analyte is captured by capture probes withinthe polymer matrix. In some embodiments, the analyte is captured bycapture probes on the surface of the polymer matrix.

In some embodiments, the analyte is released from the cell uponinteraction with a reagent. In some embodiments, the reagent is anoxidative or a reducing agent. In some embodiments, the reagent is anorganic or inorganic molecule. In some embodiments, the organic orinorganic molecule is a pharmaceutical compound or detergent. In someembodiments, the reagent is a protein. In some embodiments, the reagentis a DNA aptamer. In some embodiments, the reagent is a bead carryingbiomolecules. In some embodiments, the reagent is a biological species.In some embodiments, the biological species is a virus or cell.

In some embodiments, the analyte is released from the cell upon exposureto an energy source. In some embodiments, the energy source is UV lightfor lysing cells. In some embodiments, the energy source is visiblelight for lysing cells. In some embodiments, the UV light is used toactivate a photoactivated detergent and lyse the cell. In someembodiments, the visible light is used to activate a photoactivateddetergent and lyse the cell.

In some embodiments, the method further comprises identifying theanalyte or a component thereof. In some embodiments, the analyte is anucleic acid, amino acid, intracellular protein, surface protein,secreted protein, exosome, metabolite or lipid, or any combinationthereof. In some embodiments, the analyte is a nucleic acid molecule,and the identifying comprises sequencing the nucleic acid molecule or aderivative thereof.

In some embodiments, the method further comprises, measuring a qualityof the analyte or a component thereof. In some embodiments, the qualityis the shape or size of the analyte or component thereof. In someembodiments, the method further comprises performing one or morefunctional assays to analyze the cell or component thereof to assesscell viability, cell morphology, cell secretions, cell responses,intercellular interactions, or any combination thereof. In someembodiments, the one or more functional assays is a colorimetric assayor fluorescent assay. In some embodiments, one or more functional assaysare performed using bright-field phase contrast or fluorescent imagingof the analyte.

In some embodiments, the method further comprises performing one or moreomics assays to characterize and quantify the cell or component thereof.In some embodiments, one or more omics assays is a proteomic,transcriptomic, genomic, or epigenomic assay, or any combinationthereof. In some embodiments, the one or more omics assays is amulti-omic assay.

Another aspect of the present disclosure provides method for processingan analyte, comprising: providing a fluidic device comprising theanalyte and one or more polymer precursors; and configuring a digitalmicromirror device to direct a unit of energy generated from the energysource to a discrete area of the fluidic device to generate a polymermatrix from said one or more polymer precursors within the fluidicdevice, wherein the polymer matrix comprises or encapsulates theanalyte.

In some embodiments, the fluidic device comprises a flow channel(sometimes referred to herein as a “channel”). In some embodiments, thefluidic device comprises an open configuration. In some embodiments, thefluidic device contains one or more discrete locations wherein the oneor more discrete locations are not in fluidic communication with anotherdiscrete location. In some embodiments, the one or more discretelocations are one or more well plates. In some embodiments, the one ormore discrete locations are open at the top. In some embodiments, theone or more discrete locations comprise the analyte.

In some embodiments, the discrete area is adjacent to or surrounding theanalyte. In some embodiments, the discrete area is adjustable accordingto the analyte size, the analyte shape, or other properties of theanalyte. In some embodiments, the discrete area is identified optically.In some embodiments, a detector is configured to detect the location ofsaid analyte within the fluidic device. In some embodiments, thedetector configured to detect the location of said analyte within saidfluidic device is a microscopic objective for imaging the fluidic devicevia an image of the fluidic channel. In some embodiments, an algorithmis used to determine where the analytes or biological components arelocated based on the imaging. In some embodiments, the imaging isbright-field imaging, phase-contrast imaging, or fluorescence imaging,or any combination thereof. In some embodiments, algorithm is asupervised, a self-supervised, or an unsupervised learning algorithm. Insome embodiments, the objective is coupled to the energy source inoptical communication with the fluidic device. In some embodiments,based on information extracted by an algorithm from an image of thefluidic channel, the digital micromirror device to direct the unit ofenergy to the fluidic device.

In some embodiments, the method further comprises introducing one ormore reagents to the polymer matrix that react with the analyte. In someembodiments, the one or more reagents flow through a membrane. In someembodiments, the membrane is semi-permeable. In some embodiments, themembrane comprises pores. In some embodiments, the pores are less than10 um. In some embodiments, the one or more reagents is an enzyme,oligonucleotide, primer, or any combination thereof. In someembodiments, the one or more reagents is a lysis reagent. In someembodiments, the one or more reagents is a nucleic acid denaturationreagent. In some embodiments, the one or more reagents degrades thepolymer matrix.

In some embodiments, the analyte is a component of a cell. In someembodiments, the cell component is a small molecule composed of nucleicacids, amino acids, intracellular proteins, surface proteins, secretedproteins, exosomes, metabolites or lipids, or any combination thereof.In some embodiments, the analyte is captured by capture probes withinthe polymer matrix. In some embodiments, the analyte is captured bycapture probes on the surface of the polymer matrix.

In some embodiments, the analyte is released from the cell uponinteraction with a reagent. In some embodiments, the reagent is anoxidative or a reducing agent. In some embodiments, the reagent is anorganic or inorganic molecule. In some embodiments, the organic orinorganic molecule is a pharmaceutical compound or detergent. In someembodiments, the reagent is a protein. In some embodiments, the reagentis a DNA aptamer. In some embodiments, the reagent is a bead carryingbiomolecules. In some embodiments, the reagent is a biological species.In some embodiments, the biological species is a virus or cell.

In some embodiments, the analyte is released from the cell upon exposureto an energy source. In some embodiments, the energy source is UV lightfor lysing cells. In some embodiments, the energy source is visiblelight for lysing cells. In some embodiments, the UV light is used toactivate a photoactivated detergent and lyse the cell. In someembodiments, the visible light is used to activate a photoactivateddetergent and lyse the cell.

In some embodiments, the method further comprises identifying theanalyte or a derivative thereof. In some embodiments, the analyte is anucleic acid molecule, and the identifying comprises sequencing thenucleic acid molecule or a derivative thereof. In some embodiments, themethod further comprises measuring a quality of the analyte. In someembodiments, the quality is the shape or size of the analyte.

In some embodiments, the method further comprises performing one or morefunctional assays to analyze the analyte to assess cell viability, cellmorphology, cell secretions, cell responses, intercellular interactions,or any combination thereof. In some embodiments, the one or morefunctional assays is a colorimetric assay or fluorescent assay. In someembodiments, the one or more functional assays is performed usingbright-field phase contrast or fluorescent imaging of the analyte.

In some embodiments, the method further comprises performing one or moreomics assays to characterize and quantify the analyte or a componentthereof. In some embodiments, the one or more omics assays is aproteomic, transcriptomic, genomic, or epigenomic assay, or anycombination thereof. In some embodiments, the one or more omics assaysis a multi-omic assay.

Another aspect of the present disclosure provides a method forprocessing an analyte, comprising: providing a fluidic device comprisingthe analyte and one or more polymer precursors; and using the one ormore polymer precursors to generate a polymer matrix from said one ormore polymer precursors within the fluidic device, wherein the polymermatrix comprises the analyte, and wherein the generation of the polymermatrix is performed in absence of a physical photomask.

In some embodiments, the fluidic device comprises a flow channel. Insome embodiments, the fluidic device comprises an open configuration. Insome embodiments, the fluidic device contains one or more discretelocations wherein the one or more discrete locations is not in fluidiccommunication with another discrete location. In some embodiments, theone or more discrete locations are one or more well plates. In someembodiments, the one or more discrete locations are open at the top. Insome embodiments, the one or more discrete locations comprise theanalyte.

In some embodiments, the fluidic device further comprises one or moremonomers. In some embodiments, the fluidic device further comprises aspatial energy modulating element. In some embodiments, the fluidicdevice comprises a surface having a capture probe immobilized thereto,wherein the capture probe couples to the analyte to immobilize theanalyte to the surface. In some embodiments, the polymer matrix isgenerated adjacent to or surrounding the analyte to immobilize theanalyte. In some embodiments, the polymer matrix forms a hydrogel. Insome embodiments, the capture probe comprises one or more functionalgroups capable of interacting with the analyte. In some embodiments, theone or more functional groups comprise a complimentary DNA sequence totarget DNA or RNA.

In some embodiments, the generation of a polymer matrix within saidfluidic device comprises exposing the one or more polymer precursors toan energy source. In some embodiments, the energy source is a lightgenerating device. In some embodiments, the light generating devicegenerates light at 350 nm to 800 nm. In some embodiments, the lightgenerating device generates light at 350 nm to 600 nm. In someembodiments, the light generating device generates light at 350 nm to450 nm. In some embodiments, the light generating device generates UVlight. In some embodiments, the generation of a polymer matrix withinsaid fluidic device is performed using a spatial light modulator (SLM).In some embodiments, the SLM is a digital micromirror device (DMD). Insome embodiments, the SLM is a laser beam steered using a galvanometer.In some embodiments, the SLM is liquid-crystal based.

In some embodiments, the polymer matrix is generated in a discrete areaof the fluidic device. In some embodiments, the discrete area isadjacent to or surrounding the analyte. In some embodiments, the area ofthe discrete area is less than the area of the fluidic device. In someembodiments, the analyte is captured within the discrete area. In someembodiments, the size and shape of the discrete area is adjustableaccording to the analyte size, the analyte shape, or other properties ofthe analyte. In some embodiments, an algorithm is used to determine theshape and size of the discrete area. In some embodiments, the algorithmis a supervised, a self-supervised, or an unsupervised learningalgorithm.

In some embodiments, the discrete area is optically identified. In someembodiments, a detector is configured to detect the location of saidanalyte within said fluidic device. In some embodiments, the detectorconfigured to detect the location of the analyte within the fluidicdevice is a microscope objective for imaging the fluidic device. In someembodiments, an algorithm is used to determine where the analyte islocated based on the imaging. In some embodiments, the imaging isbright-field imaging, phase-contrast imaging, or fluorescence imaging,or any combination thereof. In some embodiments, the algorithm is asupervised, a self-supervised, or an unsupervised learning algorithm. Insome embodiments, the objective is coupled to an energy source to emitenergy to the discrete area in the fluidic device.

In some embodiments, the method further comprises introducing one ormore reagents to the polymer matrix that react with the analyte. In someembodiments, the one or more reagents flow through a membrane. In someembodiments, the membrane is semi-permeable. In some embodiments, themembrane comprises pores. In some embodiments, the pores are less than10 um. In some embodiments, the one or more reagents is an enzyme, adrug molecule, oligonucleotide, primer, or any combination thereof. Insome embodiments, the one or more reagents is a lysis reagent. In someembodiments, the one or more reagents is a nucleic acid denaturationreagent. In some embodiments, the one or more reagents degrades thepolymer matrix.

In some embodiments, the analyte is a component of a cell. In someembodiments, the analyte is a small molecule composed of nucleic acids,amino acids, intracellular proteins, surface proteins, secretedproteins, exosomes, metabolites or lipids, or any combination thereof.In some embodiments, the analyte is captured by capture probes withinthe polymer matrix. In some embodiments, the analyte is captured bycapture probes on the surface of the polymer matrix.

In some embodiments, the analyte is released from the cell uponinteraction with a reagent. In some embodiments, the reagent is anoxidative or a reducing agent. In some embodiments, the reagent is anorganic or inorganic molecule. In some embodiments, the organic orinorganic molecule is a pharmaceutical compound or detergent. In someembodiments, the reagent is a protein. In some embodiments, the reagentis a DNA aptamer. In some embodiments, the reagent is a bead carryingbiomolecules. In some embodiments, the reagent is a biological species.In some embodiments, the biological species is a virus or cell.

In some embodiments, the analyte is released from the cell upon exposureto an energy source. In some embodiments, the energy source is UV lightfor lysing cells. In some embodiments, the energy source is visiblelight for lysing cells. In some embodiments, the UV light is used toactivate a photoactivated detergent and lyse the cell. In someembodiments, the visible light is used to activate a photoactivateddetergent and lyse the cell.

In some embodiments, the method further comprises identifying theanalyte or a derivative thereof. In some embodiments, the analyte is anucleic acid molecule, and the identifying comprises sequencing thenucleic acid molecule or a derivative thereof. In some embodiments, themethod further comprises measuring a quality of the analyte. In someembodiments, the quality is the shape or size of the analyte.

In some embodiments, the method further comprises performing one or morefunctional assays to analyze the analyte to assess cell viability, cellmorphology, cell secretions, cell responses, intercellular interactions,or any combination thereof. In some embodiments, the one or morefunctional assays is a colorimetric assay or fluorescent assay. In someembodiments, the one or more functional assays is performed usingbright-field phase contrast or fluorescent imaging of the analyte.

In some embodiments, the method further comprises performing one or moreomics assays to characterize and quantify the analyte or a componentthereof. In some embodiments, the one or more omics assays is aproteomic, transcriptomic, genomic, or epigenomic assay, or anycombination thereof. In some embodiments, the one or more omics assaysis a multi-omic assay.

Another aspect of the present disclosure provides a system comprising afluidic device containing one or more biological components and one ormore polymer precursors. The system may further comprise at least oneenergy source in communication with the fluidic device. In someembodiments, the at least one energy source supplies energy to thefluidic device to cause the one or more polymer precursors to form atleast one polymer matrix on or adjacent to the biological component.

In some embodiments, the fluidic device comprises a channel disposedtherethrough. In some embodiments, a first surface is disposed along aportion of the channel, and a second surface is disposed opposite of thefirst surface. In some embodiments, the fluidic device comprises achamber disposed therein. In some embodiments, a first surface isdisposed along a portion of the chamber, and a second surface isdisposed opposite of the first surface. In some embodiments, the firstsurface is a lower surface. In some embodiments, the second surface isan upper surface. In some embodiments, the fluidic device furthercomprises one or more capture elements immobilizing at least one of theone or more biological components at a location adjacent to the firstsurface forming an immobilized biological component. In someembodiments, the first surface is disposed adjacent to the energysource. In some embodiments, the energy source is a light source. Insome embodiments, the energy source is an array of electrodes. In someembodiments, the energy source supplies electrochemical energy to theone or more polymer precursors to form an array of polymer matrices. Insome embodiments, at least two of the one or more polymer precursors arecoupled to the first surface forming a pattern on the first surface.

In some embodiments, the at least one polymer matrix is formed on oradjacent to the pattern. In some embodiments, the at least one polymermatrix is coupled to the first surface. In some embodiments, the atleast one polymer matrix extends from the first surface to the secondsurface such that the at least one polymer matrix surrounds at least aportion of the immobilized biological component.

In some embodiments, the at least one energy source is in at least oneof optical communication, electrochemical communication, electromagneticcommunication, thermal communication, or microwave communication withthe fluidic device. In some embodiments, the at least one energy sourcecomprises a light generating device, a heat generating device, anelectrochemical generating device, an electrode, or a microwave device.In some embodiments, the system further comprises a photolithographicdevice or a digital micromirror device (DMD) configured to control aspatial distribution of the energy from the energy source.

In some embodiments, the one or more capture elements comprise aphysical trap, a geometric trap, a well, an electrochemical trap, achemical affinity trap, one or more magnetic particles, anelectrophoretic trap, a dielectrophoretic trap, or a combinationthereof. In some embodiments, the chemical affinity trap comprisesstreptavidin, an antibody, or a combination thereof. In someembodiments, the physical trap comprises a polymer matrix. In someembodiments, the polymer matrix comprises a hydrogel. In someembodiments, the electrochemical trap comprises a gold electrode, aplatinum electrode, an indium tin oxide (ITO) electrode, or othersuitable electrochemical trap. In some embodiments, the one or morecapture elements are disposed in a pattern on the first surface. In someembodiments, the one or more capture elements comprises a well. In someembodiments, the well is from 1 μm (micrometer) to 50 μm in diameter. Insome embodiments, the well is from 0.1 μm to 100 μm in depth.

In some embodiments, the one or more biological components is aplurality of biological components. In some embodiments, the pluralityof biological components is coupled to the one or more capture elements.In some embodiments, the fluidic device is a microfluidic device or ananofluidic device. In some embodiments, the fluidic device is used fornucleic acid sequencing. In some embodiments, the nucleic acidsequencing comprises next-generation sequencing, short-read sequencing,nanopore sequencing, sequencing by synthesis, sequencing by in-situhybridization, or an optical readout.

In some embodiments, the one or more biological components comprise acell, a cell lysate, a nucleic acid, a microbiome, a protein, a mixtureof cells, a spatially-linked biological component, or a metabolite. Insome embodiments, the mixture of cells comprises a first cell type and asecond cell type. In some embodiments, the first cell type is differentthan the second cell type. In some embodiments, the cell is an animalcell (e.g., a human cell), a plant cell, a fungal cell, or a bacterialcell. In some embodiments, the one or more biological componentscomprise a tumor spheroid or a spatially linked biological sample.

In some embodiments, the nucleic acid is DNA of 100 base pairs orgreater or RNA of 50 bases or greater. In some embodiments, the celllysate comprises DNA from 50 bp (base pairs) to 100 Gbp (giga basepairs) or RNA from 50 bp to 100 kbp (kilo base pairs). In someembodiments, the at least one polymer matrix comprises a hydrogel. Insome embodiments, the fluidic device further comprises one or morepolymer precursors. In some embodiments, the one or more polymerprecursors comprise hydrogel precursors. In some embodiments, the atleast one polymer matrix inhibits passage of the immobilized biologicalcomponent. In some embodiments, the at least one polymer matrix forms apolymer matrix wall extending from the first surface to the secondsurface to form a chamber within the channel. For example, such chambermay comprise a cylinder shell or a polygon shell, comprising an innerspace, or interior and a polymer matrix wall. In some embodiments, suchchambers have annular-like cross-sections. As used herein, the term“annular-like cross-section” means a cross-section topologicallyequivalent to an annulus. In some embodiments, the inner space, orinterior, of a chamber has an inner diameter from 1 μm to 500 μm and avolume in the range of from 1 picoliter to 200 nanoliters, or from 100picoliters to 100 nanoliters, or from 100 picoliters to 10 nanoliters.In some embodiments, the polymer matrix wall has a thickness of at least1 μm (micrometer). In some embodiments, a polymer matrix wall having anannular-like cross-section has an aspect ratio (i.e., height/width) of 1or less. In some embodiments, aspect ratio and polymer matrix wallthickness are selected to maximize chamber stability against forces,such as reagent flow through the channel, washings, and the like. Insome embodiments, the at least one polymer matrix wall is a hydrogelwall. In some embodiments, the at least one polymer matrix isdegradable. In some embodiments, the degradation of the at least onepolymer matrix is “on demand.”

In some embodiments, on demand degradation may be implemented usingpolymer precursors that permit photo-crosslinking and photo-degradation,for example, using different wavelengths for crosslinking and fordegradation. For example, Eosin Y may be used for radical polymerizationat defined regions using 500 nm wavelength, after which illumination at380 nm can be used to cleave the cross linker. In other embodiments,photo-caged hydrogel cleaving reagents may be included in the formationof polymer matrix walls. For example, acid labile crosslinkers (such asesters, or the like) can be used to create the hydrogel and then UVlight can be used to generate local acidic conditions which, in turn,degrades the hydrogel.

In some embodiments, the at least one polymer matrix is degradable by atleast one of: (i) contacting the at least one polymer matrix with acleaving reagent; (ii) heating the at least one polymer matrix to atleast 90° C.; or (iii) exposing the at least one polymer matrix to awavelength of light that cleaves a photo-cleavable crosslinker thatcrosslinks the polymer of the at least one polymer matrix. In someembodiments, the at least one polymer matrix comprises a hydrogel. Insome embodiments, the cleaving reagent degrades the hydrogel. In someembodiments, the cleaving reagent comprises a reducing agent, anoxidative agent, an enzyme, a pH based cleaving reagent, or acombination thereof. In some embodiments, the cleaving reagent comprisesdithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP),tris(3-hydroxy propyl)phosphine (THP), or a combination thereof.

In some embodiments, the at least one polymer matrix allows passage of areagent. In some embodiments, the at least one polymer matrix comprisespores. In some embodiments, an average size of the pores is modulatedusing a chemical reagent, by applying heat, applying electricity,applying light, or a combination thereof. In some embodiments, thereagent comprises at least one of enzymes, chemicals, oligonucleotides,or primers having a size of less than 50 base pairs.

In some embodiments, the reagent comprises lysozyme, proteinase K,random hexamers, polymerase, transposase, ligase, catalyzing enzyme,deoxynucleotide triphosphates, buffers, cell culture media, or divalentcations. In some embodiments, the at least one polymer matrix comprisespores that are sized to allow diffusion of a reagent through the atleast one polymer matrix but are too small to allow DNA or RNA foranalysis to traverse the pores. In some embodiments, DNA or RNA retainedhave lengths that are sequencable using conventionalsequencing-by-synthesis techniques. For example, such DNA or RNAcomprise at least 50 nucleotides, or in some embodiments, at least 100nucleotides. In some embodiments, the at least one polymer matrixcomprises a hydrogel. In some embodiments, the hydrogel comprisespolyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide,N,N′-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylicacid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate)(PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, bisacrylamide, diacrylate, diallylamine,triallylamine, divinyl sulfone, diethyleneglycol diallyl ether,ethyleneglycol diacrylate, polymethyleneglycol diacrylate,polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate,ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritoltetraacrylate, or combinations or mixtures thereof. In some embodiments,the hydrogel comprises an enzymatically degradable hydrogel,PEG-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),or PEG/PPO. In some embodiments, the following precursors andcrosslinker may be used to form chambers with degradable polymer matrix(hydrogel) walls. Polymer precursors may be formed by using any hydrogelprecursor and crosslinkers of Table 1A (columns 1 and 3, respectively).The resulting polymer matrices may be degraded with the indicateddegradation agents in Table 1A (column 4).

TABLE 1A Precursors Hydrogels Crosslinkers Degradation Agents AcrylamidePolyacrylamide Bis-acryloyl cystamine (structure 1) DTT/TCEP/THPPEG-based PEG Bis(2-methacryloly)oxyethyl disulfide DTT/TCEP/THPacryloyl (structure 2) Dextran-based DextranN,N′-(1,2-Dihydroxylethylene)bis- NaIO4 acryloyl acrylamide structure(3) Polysaccharide- Polysaccharide Structure 4 NaOH, ethanolamine baseacryloyl DTT/TCEP/THP Gelatin-base Gelatin Structure 5 NaOH,ethanolamine, acryloyl nucleophilic bases Structure 6 NaOH, alkali,organic bases Structure 7 Acid

TABLE 1B Structure Number Structure 1

2

3

4

5

6

7

In some embodiments, the first surface, the second surface, or bothcomprise one or more barcodes. In some embodiments, the one or morebarcodes comprise an identifier to identify a source of the one or morebiological components. In some embodiments, the source comprises aspecimen from which the one or more biological components are collected.In some embodiments, the source comprises a physiological or ananatomical source from which the one or more biological components arecollected. In some embodiments, the anatomical source comprises an organof a subject. In some embodiments, the subject is a human. In someembodiments, the one or more barcodes are configured to bind the one ormore biological components, or a molecule made by the one or morebiological components.

In some embodiments, the first surface, the second surface, or bothcomprise one or more compounds configured to bind the one or morebiological components. In some embodiments, the first surface or thesecond surface is functionalized with a surface polymer. In someembodiments, the surface polymer is functionalized with anoligonucleotide, an antibody, a cytokine, a chemokine, a protein, anantibody derivative, an antibody fragment, a carbohydrate, a toxin, anaptamer, or any combination thereof. In some embodiments, a surface ofthe polymer matrix is functionalized with an oligonucleotide, anantibody, a cytokine, a chemokine, a protein, an antibody derivative, anantibody fragment, a carbohydrate, a toxin, an aptamer, or anycombination thereof. In some embodiments, the surface polymer comprisespolyethylene glycol (PEG), a silane polymer, pyridinecarboxaldehyde(PCA), an acrylamide, an agarose, or a combination thereof.

In some embodiments, the system further comprises a detector foridentifying the one or more of the biological components, the one ormore barcodes, or a combination thereof. In some embodiments, thedetector comprises a camera (fluorescent camera).

In some embodiments, the system further comprises a stage that holds thefluidic device. In some embodiments, the system further comprises asequencing device for obtaining sequencing data. In some embodiments,the sequencing data is generated using next-generation sequencing,short-read sequencing, nanopore sequencing, sequencing by synthesis,sequencing by in-situ hybridization, or an optical readout.

In some embodiments, the system further comprises a spatial energymodulating element to selectively supply the energy to the fluidicdevice. In some embodiments, the spatial energy modulating element isgenerated using the detector identifying the position of the at leastone biological component. In some embodiments, the spatial energymodulating element comprises a physical photomask, a virtual photomask,a physical electrode distribution pattern, or a virtual electrodedistribution pattern. In some embodiments, the spatial energy modulatingelement comprises a photolithographic mask or a digital micromirrordevice (DMD) mask.

Another aspect of the present disclosure provides a method of analyzinga biological component. The method may comprise (a) introducing one ormore biological components into a fluidic device; (b) disposing a firstportion of the one or more biological components adjacent a firstsurface of the fluidic device; and (c) forming one or more polymermatrices adjacent a first portion of the first surface to localize atleast one of the one or more biological components to the first portion.

In some embodiments, the method further comprises: (d) agitating the oneor more biological components within the fluidic device; (e) disposing asecond portion of the one or more biological components adjacent thefirst surface of the fluidic device; and (f) forming one or more polymermatrices adjacent a second portion of the first surface to immobilize atleast one of the one or more biological component of the second portion.The step of agitating in this and other embodiments may prevent ordisrupt aggregation of biological components, such as, biological cells.Agitating may also induce a more uniform distribution of biologicalcomponents on a first surface within a channel.

In some embodiments, the method further comprises identifying a positionof at least one of the one or more biological components such that atleast one energy source supplies energy to the fluidic device to formthe one or more polymer matrices on or adjacent the identified position.

Another aspect of the present disclosure provides a method of analyzinga biological component. The method may comprise: (a) introducing thebiological component into a fluidic device; (b) coupling the biologicalcomponent to one or more capture elements disposed on a first surface orsecond surface of the fluidic device to yield a coupled biologicalcomponent; and (c) forming a polymer matrix on or adjacent to thecoupled biological component.

In some embodiments, the method further comprises introducing one ormore polymer precursors into the fluidic device. In some embodiments,forming the polymer matrix comprises supplying energy to the fluidicdevice to form the polymer matrix.

In some embodiments, the energy is selectively supplied to one or moreportions of the fluidic device. In some embodiments, the method furthercomprises a spatial energy modulating element to selectively supply theenergy to the fluidic device. In some embodiments, the spatial energymodulating element comprises a physical photomask, a virtual photomask,a physical electrode distribution pattern, or a virtual electrodedistribution pattern.

In some embodiments, the spatial energy modulating element comprises aphotolithographic mask or a digital micromirror device (DMD) mask. Insome embodiments, the energy is supplied via a light energy source, aheat energy source, an electrochemical energy source, or anelectromagnetic energy source.

In some embodiments, the polymer matrix is coupled to the first surface.In some embodiments, the energy forms the polymer matrix around aportion of the coupled biological component. In some embodiments, atleast a portion of the biological component is encapsulated by thepolymer matrix. In some embodiments, an entirety of the biologicalcomponent is encapsulated by the polymer matrix.

In some embodiments, the method further comprises coupling a firstbiological component to a first capture element to form a first analysischamber and coupling a second biological component to a second captureelement to form a second analysis chamber. In some embodiments, thefirst analysis chamber is adjacent to the second analysis chamber. Insome embodiments, the first analysis chamber is disposed from 5micrometer (μm) to 1,000 μm away from the second analysis chamber.

In some embodiments, the method further comprises analyzing the firstbiological component in the first analysis chamber and analyzing thesecond biological component in the second analysis chamber. In someembodiments, the method further comprises actuating a first reaction inthe first biological component and actuating a second reaction in thesecond biological component. In some embodiments, the first reaction andthe second reaction are different. In some embodiments, the methodfurther comprises actuating a third reaction in the first biologicalcomponent and actuating a fourth reaction in the second biologicalcomponent. In some embodiments, the third reaction and the fourthreaction are different.

In some embodiments, the method further comprises obtaining a genome,transcriptome, proteome, epigenome, methylome, secretome, or metabolomeof the coupled biological component.

In some embodiments, the proteome comprises secreted proteins, surfaceproteins, or a combination thereof. In some embodiments, thetranscriptome is a substantially full-length transcriptome. In someembodiments, the transcriptome is a full-length transcriptome. In someembodiments, the method further comprises sequencing at least onenucleic acid of the biological component. In some embodiments, thesequencing does not comprise amplification of a sequencing library. Insome embodiments, the nucleic acid library from the biological componentis sequenced within a same chamber. In some embodiments, the methodfurther comprises coupling a barcode to the biological component or amolecule produced by the biological component.

In some embodiments, the method further comprises exposing thebiological component or the coupled biological component to an analyte.In some embodiments, the biological component comprises one or moremicrobes. In some embodiments, the analyte comprises an antimicrobialagent or a microbial growth promoting agent. In some embodiments, themethod further comprises screening the one or more microbes forsusceptibility to the antimicrobial agent. In some embodiments, theanalyte comprises a pharmaceutical agent.

In some embodiments, the method further comprises screening an effect ofthe pharmaceutical agent on the biological component. In someembodiments, the method further comprises screening the biologicalcomponent for production of a target molecule.

In some embodiments, the target molecule comprises at least one of anantibody, a cytokine, a chemokine, a protein, an antibody derivative, anantibody fragment, a carbohydrate, a toxin, or an aptamer.

In some embodiments, the method further comprises forming the polymermatrix around the biological component such that the biologicalcomponent is disposed within a structure formed by the polymer matrix.

In some embodiments, the method further comprises analyzing a localparameter in the first analysis chamber or the second analysis chamber.In some embodiments, a level of the local parameter in the firstanalysis chamber is different from a level of the local parameter in thesecond analysis chamber. In some embodiments, the local parametercomprises a pH, an oxygen concentration, or a CO2 concentration.

In some embodiments, the one or more capture elements comprise a polymermatrix. In some embodiments, the polymer matrix comprises a hydrogel.

Another aspect of the present disclosure provides a method of obtaininga transcriptome of a biological component. The method may comprise (a)forming a polymer matrix on or adjacent to the biological component toform an analysis chamber; and (b) performing one or more reactions inthe analysis chamber to obtain the transcriptome of the biologicalcomponent. In some embodiments, the biological component remains in orsubstantially in the analysis chamber during performance of the one ormore reactions.

In some embodiments, the method further comprises coupling thebiological component to a capture element disposed in a fluidic deviceto yield a coupled biological component. In some embodiments, the methodfurther comprises providing energy from an energy source to the fluidicdevice to form the polymer matrix. In some embodiments, the energy isprovided selectively using a spatial energy modulating element. In someembodiments, the spatial energy modulating element is generated based ona location of the biological component. In some embodiments, the spatialenergy modulating element comprises a physical photomask, a virtualphotomask, a physical electrode distribution pattern, a virtualelectrode distribution pattern, a photolithographic mask, or a digitalmicromirror device (DMD) mask.

In some embodiments, the biological component comprises RNA. In someembodiments, the RNA is from 50 bases to 100 kb (kilobase bases). Insome embodiments, the polymer matrix comprises pores that are sized toallow diffusion of a reagent through the polymer matrix but are toosmall to allow the RNA to traverse the pores. In some embodiments, theone or more reactions comprise RNA sequencing.

Another aspect of the present disclosure provides a method of analyzingtwo or more biological components. The method may comprise (a)introducing a first biological component and a second biologicalcomponent into a fluidic device; (b) forming a polymer matrix on oradjacent to the first biological component to form a first analysischamber and forming a polymer matrix on or adjacent to the secondbiological component to form a second analysis chamber; and (c)analyzing one or more features of the first biological component and thesecond biological component. In some embodiments, the first analysischamber is adjacent to the second analysis chamber in the fluidicdevice. In some embodiments, the one or more features comprise a firstfeature and a second feature. In some embodiments, (c) comprisesanalyzing the first feature and the second feature of the firstbiological component in the first analysis chamber.

In some embodiments, the first biological component remains in the firstanalysis chamber between analysis of each of the first feature and thesecond feature. In some embodiments, the one or more features comprisesa response to an analyte, a response to a pharmaceutical agent, aresponse to an antimicrobial agent, production of a target compound,production of a target molecule, production of a nucleic acid, orproduction of a protein. In some embodiments, the first biologicalcomponent is in biological communication with the second biologicalcomponent. In some embodiments, the biological communication generates abiological response in the first biological component or in the secondbiological component. In some embodiments, the biological communicationcomprises a molecule comprising a protein, a nucleic acid, a cytokine, achemokine, or a combination thereof, generated by the first biologicalcomponent or by the second biological component.

Another aspect of the present disclosure provides a method foridentifying a nucleic acid molecule. The method may comprise providing apolymer matrix comprising the nucleic acid molecule, and detecting thenucleic acid molecule in absence of nucleic acid amplification.

In some embodiments, the nucleic acid molecule is a deoxyribonucleicacid (DNA) molecule.

In some embodiments, the polymer matrix forms a chamber localizing thenucleic acid. In some embodiments, the chamber is formed on demand. Insome embodiments, the polymer matrix is degraded on demand. In someembodiments, the DNA is 100 base pairs or greater. In some embodiments,the nucleic acid is a ribonucleic acid molecule (RNA). In someembodiments, the RNA is 50 nucleotides or greater. In some embodiments,the method further comprises generating a nucleic acid library from thebiological component within the chamber. In some embodiments, thenucleic acid library is sequenced within the chamber.

Another aspect of the present disclosure provides method for processinga biological component. The method may comprise determining a genomesequence, a transcriptome, a proteome, or an epigenome in absence ofnucleic acid amplification. In some embodiments, the processing isperformed in a single microfluidic device. In some embodiments, the cellis at least partially within a polymer matrix. In some embodiments, thepolymer matrix is degraded on demand. In some embodiments, the methodfurther comprises determining methylation in the cell.

Another aspect of the present disclosure provides a method comprisingidentifying a plurality of nucleic acid molecules of a plurality ofcells without barcoding individual nucleic acid molecules of theplurality of nucleic acid molecules. In some embodiments, extracting theplurality of nucleic acid molecules and identifying are performed in asingle microfluidic device. In some embodiments, the identifyingcomprises sequencing. In some embodiments, the method further comprisesforming a polymer matrix on or adjacent to individual cells of theplurality of cells such that the individual cells are separated from oneanother. In some embodiments, the method further comprises extractingthe individual nucleic acid molecules the individual cells. In someembodiments, the sequencing comprises sequencing the individual nucleicacid molecules within the polymer matrix. In some embodiments, thesequencing comprises next-generation sequencing, short-read sequencing,nanopore sequencing, sequencing by synthesis, sequencing by in-situhybridization, or an optical readout.

Another aspect of the present disclosure provides a method comprising(a) providing a plurality of nucleic acid molecules within a pluralityof matrices; and (b) sequencing the plurality of nucleic acid moleculeswhile the plurality of nucleic acid molecules is within the plurality ofmatrices. In some embodiments, individual nucleic acid molecules of theplurality of nucleic acid molecules are from different cells. In someembodiments, the plurality of matrices is disposed in a fluidic device.In some embodiments, the plurality of matrices comprises a plurality ofcells. In some embodiments, the plurality of cells comprises theplurality of nucleic acid molecules. In some embodiments, the sequencingcomprises next-generation sequencing, short-read sequencing, nanoporesequencing, sequencing by synthesis, sequencing by in-situhybridization, or an optical readout.

Another aspect of the present disclosure provides a method of analyzinga biological component. The method may comprise (a) introducing one ormore biological components into a fluidic device; (b) disposing a firstportion of the one or more biological components adjacent a firstsurface of the fluidic device; and (c) forming one or more polymermatrices adjacent a first portion of the first surface to localize atleast one of the one or more biological component to the first portion.In some embodiments, the method further comprises: (d) agitating the oneor more biological components within the fluidic device; (e) disposing asecond portion of the one or more biological components adjacent thefirst surface of the fluidic device; and (f) forming one or more polymermatrices adjacent a second portion of the first surface to immobilize atleast one of the one or more biological component of the second portion.In some embodiments, the method further comprises identifying a positionof at least one of the one or more biological components such that atleast one energy source supplies energy to the fluidic device to formthe one or more polymer matrices on or adjacent the identified position.

Another aspect of the present disclosure provides a system comprising afluidic device. The fluidic device may comprise a flow channel, ananalysis channel disposed adjacent to the flow channel, a layer disposedbetween the flow channel and the analysis channel, and at least oneenergy source in communication with the fluidic device. In someembodiments, at least one flow inhibition element is disposed within theflow channel to inhibit flow of a biological component. In someembodiments, the layer comprises at least one sealable aperture disposedadjacent the at least one flow inhibition element. In some embodiments,the at least one sealable aperture is configured to allow passage of thebiological component. In some embodiments, the at least one energysource is configured to form a polymer matrix within the analysischannel.

In some embodiments, a portion of the flow channel is substantiallyparallel with a portion of the analysis channel. In some embodiments,the at least one sealable aperture is configured to transition from asealed state to an open state. In some embodiments, passage of thebiological component through the at least one sealable aperture isinhibited in the sealed state. In some embodiments, passage of thebiological component through the at least one sealable aperture isinhibited in the sealed state. In some embodiments, when the at leastone sealable aperture is in the sealed state, the at least one sealableaperture is sealed with at least one of an agarose gel, atemperature-soluble polymer, an N-isopropylacrylamide (NIPAAm) polymer,a wax compound, or an alginate.

In some embodiments, the flow channel comprises a surface disposedopposite of a flow channel surface of the layer. In some embodiments, atleast one of the at least one inhibition elements extends from thesurface toward the flow channel surface such that flow of the biologicalcomponent in the flow channel is inhibited by the at least oneinhibition element. In some embodiments, the analysis channel comprisesa surface disposed opposite of the analysis channel surface of thelayer. In some embodiments, the flow channel is removably couplable tothe analysis channel. In some embodiments, the surface of the analysischannel comprises one or more barcodes. In some embodiments, the barcodecomprises an oligonucleotide.

In some embodiments, the polymer matrix is coupled to at least one ofthe surface of the analysis channel or the analysis channel surface ofthe layer. In some embodiments, the polymer matrix extends from thesurface of the analysis channel to the analysis channel surface of thelayer such that the polymer matrix surrounds at least a portion of thebiological component.

In some embodiments, the at least one energy source is in at least oneof optical communication, electrochemical communication,electromagnetic. communication, thermal communication, or microwavecommunication with the fluidic device. In some embodiments, the at leastone energy source. comprises a light generating device, a heatgenerating device, an electrochemical generating device, an electrode,or a microwave device.

In some embodiments, the biological component comprises a plurality ofbiological components.

In some embodiments, the fluidic device is a microfluidic device or ananofluidic device. In some embodiments, the fluidic device comprises asequencing flow cell. In some embodiments, the fluidic device is usedfor nucleic acid sequencing. In some embodiments, the biologicalcomponent comprises a cell, a nucleic acid, a microbiome, a protein, acombination of cells, a spatially-linked biological component, or ametabolite. In some embodiments, the cell is an animal cell (e.g., ahuman cell), a plant cell, a fungal cell, a bacterial cell, a tumorspheroid, or a combination thereof. In some embodiments, the nucleicacid is DNA of 100 base pairs or greater or RNA of 50 bases or greater.

In some embodiments, the polymer matrix comprises a hydrogel. In someembodiments, the fluidic device further comprises one or more polymerprecursors. In some embodiments, the one or more polymer precursorscomprise hydrogel precursors. In some embodiments, the polymer matrixcomprises a polymer matrix wall having a width of at least 1 μm. In someembodiments, the polymer matrix inhibits passage of the biologicalcomponent. In some embodiments, the polymer matrix wall is a hydrogelwall. In some embodiments, the polymer matrix is degradable. In someembodiments, the degradation of the polymer matrix is “on demand.” Insome embodiments, the polymer matrix is degradable by at least one of:(i) contacting the polymer matrix with a cleaving reagent; (ii) heatingthe polymer matrix to at least 90° C.; or (iii) exposing the polymermatrix to a wavelength of light that cleaves a photo-cleavablecrosslinker that crosslinks the polymer of the polymer matrix.

In some embodiments, the sealable aperture is transitioned to the openstate by at least one of: (i) contacting the sealable aperture with acleaving reagent; (ii) heating the sealable aperture to at least 90° C.;or (iii) exposing the sealable aperture to a wavelength of light thatcleaves a photo-cleavable crosslinker that crosslinks the polymer of thesealable aperture. In some embodiments, the polymer matrix comprises ahydrogel. In some embodiments, the cleaving reagent is configured todegrade the polymer matrix. In some embodiments, the cleaving reagentcomprises a reducing agent, an oxidative agent, an enzyme, a pH basedcleaving reagent, or a combination thereof. In some embodiments, thecleaving reagent comprises dithiothreitol (DTT),tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxy propyl)phosphine(THP), or a combination thereof.

In some embodiments, the polymer matrix allows passage of a reagent. Insome embodiments, the polymer matrix comprises pores. In someembodiments, a size of the pores are controlled by changing acomposition of the one or more polymer precursors, the at least oneenergy source, or a combination thereof.

In some embodiments, the reagent comprises at least one of enzymes,chemicals, oligonucleotides, or primers having a size of less than 50base pairs. In some embodiments, the reagent comprises lysozyme,proteinase K, random hexamers, polymerase, transposase, ligase,catalyzing enzyme, deoxynucleotide triphosphates, buffers, cell culturemedia, or divalent cations.

In some embodiments, the polymer matrix comprises pores that are sizedto allow diffusion of a reagent through the matrix but are too small toallow DNA or RNA to traverse the pores. In some embodiments, the polymermatrix comprises a hydrogel. In some embodiments, the hydrogel comprisespolyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide,N,N′-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylicacid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate)(PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, bisacrylamide, diacrylate, diallylamine,triallylamine, divinyl sulfone, diethyleneglycol diallyl ether,ethyleneglycol diacrylate, polymethyleneglycol diacrylate,polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate,ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritoltetraacrylate, or combinations or mixtures thereof. In some embodiments,the hydrogel comprises an enzymatically degradable hydrogel,PEG-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),or PEG/PPO.

In some embodiments, the surface comprises one or more barcodes. In someembodiments, the surface of the analysis channel comprises one or morecompounds configured to bind the biological component. In someembodiments, the surface of the analysis channel is functionalized witha surface polymer. In some embodiments, the surface polymer isfunctionalized with an oligonucleotide, an antibody, a cytokine, achemokine, a protein, an antibody derivative, an antibody fragment, acarbohydrate, a toxin, an aptamer, or any combination thereof. In someembodiments, a surface of the polymer matrix is functionalized with anoligonucleotide, an antibody, a cytokine, a chemokine, a protein, anantibody derivative, an antibody fragment, a carbohydrate, a toxin, anaptamer, or any combination thereof. In some embodiments, the surfacepolymer comprises polyethylene glycol (PEG), a silane polymer,pyridinecarboxaldehyde (PCA), an acrylamide, an agarose, or acombination thereof.

In some embodiments, the system further comprises a detector foridentifying the one or more of the biological components, the one ormore barcodes, or a combination thereof. In some embodiments, thedetector comprises a camera. In some embodiments, the system furthercomprises a stage that holds the fluidic device. In some embodiments,the system further comprises a sequencing device for obtainingsequencing data.

In some embodiments, the system further comprises a spatial energymodulating element to selectively supply the energy to the fluidicdevice. In some embodiments, the spatial energy modulating elementcomprises a physical photomask, a virtual photomask, a physicalelectrode distribution pattern, a virtual electrode distributionpattern. In some embodiments, the spatial energy modulating elementcomprises a photolithographic mask or a digital micromirror device (DMD)mask.

Another aspect of the present disclosure provides a method of analyzinga biological component. The method may comprise (a) introducing thebiological component into a flow channel of a fluidic device; (b)inhibiting flow of the biological component adjacent to an inhibitionelement; (c) disposing the biological component from the flow channel toan analysis channel of the fluidic device; and (d) forming a polymermatrix on or adjacent to the biological component in the analysischannel either before or after the disposition of the biologicalcomponent in the analysis channel. In some embodiments, a sealableaperture is disposed adjacent to the inhibition element.

In some embodiments, prior to the disposing in (c), the sealableaperture is degraded using at least one of: (i) contacting the sealableaperture with a cleaving reagent; (ii) heating the sealable aperture toat least 90° C.; or (iii) exposing the sealable aperture to a wavelengthof light that cleaves a photo-cleavable crosslinker that crosslinks thepolymer of the sealable aperture. In some embodiments, the methodfurther comprises introducing one or more polymer precursors into thefluidic device. In some embodiments, forming the polymer matrixcomprises supplying energy to the fluidic device to form the polymermatrix. In some embodiments, the energy is selectively supplied to oneor more portions of the fluidic device. In some embodiments, the methodfurther comprises activating a spatial energy modulating element toselectively supply the energy to the fluidic device. In someembodiments, the spatial energy modulating element comprises a physicalphotomask, a virtual photomask, a physical electrode distributionpattern, or a virtual electrode distribution pattern. In someembodiments, the spatial energy modulating element comprises aphotolithographic mask or a digital micromirror device (DMD) mask. Insome embodiments, the energy is supplied via light energy source, a heatenergy source, an electrochemical energy source, or an electromagneticenergy source.

In some embodiments, the polymer matrix is coupled to a surface of theanalysis channel. In some embodiments, the energy forms the polymermatrix around a portion of the coupled biological component. In someembodiments, at least a portion of the biological component isencapsulated by the polymer matrix. In some embodiments, an entirety ofthe biological component is encapsulated by the polymer matrix.

In some embodiments, the method further comprises encapsulating a firstbiological component to form a first analysis chamber and encapsulatinga second biological component to form a second analysis chamber. In someembodiments, the first analysis chamber is adjacent to the secondanalysis chamber. In some embodiments, the first analysis chamber isdisposed from 5 micrometer (μm) to 1,000 μm away from the secondanalysis chamber.

In some embodiments, the method further comprises analyzing the firstbiological component in the first analysis chamber and analyzing thesecond biological component in the second analysis chamber. In someembodiments, the method further comprises actuating a first reaction inthe first biological component and actuating a second reaction in thesecond biological component. In some embodiments, the first reaction andthe second reaction are different. In some embodiments, the methodfurther comprises actuating a third reaction in the first biologicalcomponent and actuating a fourth reaction in the second biologicalcomponent. In some embodiments, the third reaction and the fourthreaction are different.

In some embodiments, the method further comprises obtaining a genome,transcriptome, proteome, epigenome, methylome, secretome, or metabolomeof the biological component. In some embodiments, the transcriptome is asubstantially full-length transcriptome. In some embodiments, thetranscriptome is a full-length transcriptome. In some embodiments, themethod further comprises sequencing the biological component. In someembodiments, the sequencing does not comprise amplification of asequencing library. In some embodiments, the method further comprises abarcode configured to be coupled to the biological component or amolecule produced by the biological component.

In some embodiments, the method further comprises exposing thebiological component or the first biological component to an analyte. Insome embodiments, the biological component comprises one or moremicrobes. In some embodiments, the analyte comprises an antimicrobialagent, a microbial growth promoting chemical, or a combination thereof.In some embodiments, the method further comprises screening the one ormore microbes for susceptibility to the antimicrobial agent. In someembodiments, the analyte comprises a pharmaceutical agent.

In some embodiments, the method further comprises screening an effect ofthe pharmaceutical agent on the biological component. In someembodiments, the method further comprises screening the biologicalcomponent for production of a target molecule. In some embodiments, thetarget molecule comprises at least one of an antibody, a cytokine, achemokine, a protein, an antibody derivative, an antibody fragment, acarbohydrate, a toxin, or an aptamer.

In some embodiments, the method further comprises forming the polymermatrix around the biological component such that the biologicalcomponent is disposed within a structure formed by the polymer matrix.In some embodiments, the method further comprises analyzing a localparameter in the first analysis chamber or the second analysis chamber.In some embodiments, a level of the local parameter in the firstanalysis chamber is different from a level of the local parameter in thesecond analysis chamber. In some embodiments, the local parametercomprise a pH, an oxygen concentration, or a CO2 concentration.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

In some embodiments, the invention provides a method of analyzing one ormore biological components, such as mammalian cells, of a biologicalsample comprising the following steps: (a) providing a fluidic devicehaving (i) a channel with an inlet and an outlet, the channel beingbounded by a first wall having a first surface and a second wall havinga second surface wherein the first and second walls are disposedopposite one another across the channel, (ii) a spatial energymodulating element disposed adjacent to the first wall, the spatialenergy modulating element being capable of projecting energy across thechannel with predetermined beam characteristics; (b) loading the channelwith a reaction mixture comprising a biological sample and one or morepolymer precursors; (c) synthesizing one or more chambers in the channelaround each of one or more biological components by projecting energyacross the channel such that the projected energy cross-links the one ormore polymer precursors to form polymer matrix walls of the chambers. Insome embodiments, the channel is planar in shape with width and breadthbeing large with respect to its height. The cross-section of the channelperpendicular to the direction of reagent flow is typically rectangular.Reaction mixtures or other reagents, e.g. wash solutions, and the like,are loaded into the channel through an inlet and are removed through anoutlet. In some embodiments, such reagents or mixtures may be pumpedthrough the inlet and into the channel, for example, using a syringepump. In some embodiments, an outlet may be a vent port for air from thechannel to escape whenever a reagent is loaded into the channel by wayof the inlet, thereby displacing air.

In some embodiments, the invention provides a method of analyzing one ormore biological components, such as mammalian cells, of a biologicalsample comprising the following steps: (a) providing a fluidic devicehaving (i) a channel, the channel being bounded by a first wall having afirst surface, (ii) a spatial energy modulating element disposedadjacent to the first wall, the spatial energy modulating element beingcapable of projecting energy across the first wall into the channel withpredetermined beam characteristics; (b) loading the channel with areaction mixture comprising a biological sample and one or more polymerprecursors; (c) synthesizing one or more chambers in the channel aroundeach of one or more biological components by projecting energy into thechannel such that the projected energy cross-links the one or morepolymer precursors to form polymer matrix walls of the chambers. Thisembodiment is sometimes referred to herein as an “open configuration” ofthe system of the invention. In some open configuration embodiments, thechambers comprise wells or containers which are open at the top. In someembodiments, the top of a well or container is opposite a first surface.

In some embodiments, the fluidic device of the method further comprisesa detector disposed adjacent to the second wall or opposite the firstwall from the spatial energy modulating element in open configurationembodiments. The detector is positioned so that it is capable ofdetecting optical signals from one or more biological components, suchas mammalian cells, distributed over the first surface in chambers. Insome embodiments, the first and second walls each comprise opticallytransmissive material, for example, so that a spatial energy modulatingelement may project light energy to the interior of the channel, and sothat a detector may detect optical signals, such as fluorescentemissions or reflected light from biological components. In someembodiments, the projected energy from the spatial energy modulatingelement is a light energy from a light beam. In some embodiments, thelight beam projected by the spatial energy modulating element may have acomplex cross-section that permits (in various embodiments) thesimultaneous synthesis of a plurality of chambers. Opticallytransmissive materials include, but are not limited to, glass, quartz,plastic, and like materials.

In some embodiments, the step of synthesizing chambers includespositioning the chambers so that they encapsulate the one or morebiological components based on the optical signals detected by thedetector. That is, in some embodiments, the detector is operationallyassociated with the spatial energy modulating element to selectivelyproject one or more light beams to locations where detected opticalsignals indicate the presence of biological components of interest. Insuch embodiments, the detector and spatial energy modulating element areoperationally associated so that the spatial energy modulating elementis configured to generate an energy beam having predetermined beamcharacteristics. For example, one such characteristic may be a beamcross-section which results in the biological components of interestbeing encapsulated by chambers. In such operational association, opticalsignals detected by the detector may include, but is not limited to,morphology of biological components, for example, cell morphology;motility of biological components, such as cells; interaction of onecell type with another cell type, such as binding of one cell type toanother cell type; a presence, absence or quantity of a label on thebiological component, or the like.

In some embodiments, when chambers are formed or synthesized the polymermatrix walls of the chambers extend from the first surface to the secondsurface to form chambers each having an interior. In other embodiments,a fluidic device may not have a second wall with a second surface, whichis referred to herein as an “open configuration.” In such embodiments,chambers form open cylinder or well shapes on the first surface. Theheight and thickness of the well walls are determined in part by theintensity and duration of photo-illumination by the spatial energymodulating element. In some embodiments, predetermined beamcharacteristics comprise a beam having an annular-like cross-section. Inother embodiments, predetermined beam characteristics comprise a beamhaving a cross-section comprising a plurality of annular-like shapes,which may be isolated and noncontiguous, or which may include contiguousannular-like shapes. In other embodiments, predetermined beamcharacteristics comprise a beam that generates a plurality of chamberswherein a portion of the chambers share polymer matrix walls with one ormore other chambers of the plurality, such as shown in FIG. 25B.

In some embodiments, the step of loading may further include a step ofagitating reaction mixture, for example, by vibrating, shaking oragitating the channel, to reduce aggregation of the biologicalcomponents. Such agitation may be for a few minutes, e.g. 1-30 minutes,to an hour or more, e.g. 1-2 hours.

In some embodiments, the first or second surface, usually the firstsurface, includes one or more capture elements for specificallycapturing one or more predetermined biological components of saidbiological sample, for example, selected cells, such as lymphocytes, orthe like. In some embodiments, the step of loading may includeincubating the reaction mixture containing such predetermined biologicalcomponents under conditions to permit the one or more capture elementsto capture the one or more predetermined biological components. Suchincubation may be for a few minutes to an hour or more. In someembodiments, such incubation time may be in the range of from 1 minuteto 10 hours, or from 10 minutes to 2 hours, or from 30 minutes to 2hours.

In some embodiments, the method of the invention includes removing thereaction mixture from the channel after the step of synthesizing thechambers is completed. Such removing may include washing the channelwith a buffer solution.

In some embodiments, the method of the invention, after chambers areformed, includes a step of loading said channel analytical assayreagents for determining one of more characteristics of said biologicalcomponents encapsulated in said chambers, wherein the analytical assayreagents are capable of passing through said polymer matrix walls andare capable of generating one or more optical signals indicative of theone or more characteristics. A wide variety of analytical assays may beperformed, as indicated else where in the present application, includingRNA or DNA identification and/or sequencing, protein identificationand/or quantification, omics assays, or the like.

In some embodiments, the polymer matrix walls of chambers are degradableby treating the chambers with a degradation agent. In such embodiments,the method of the invention may include the further steps of (a)identifying one or more of said chambers having said biologicalcomponents with selected characteristics based on said one or moreoptical signals generated from operation of the analytical assayreagents; (b) loading the channel with a second reaction mixturecomprising second polymer precursors, wherein the second polymerprecursors are capable of forming second polymer matrix walls which arenondegradable for at least one degradation agent; and (c) synthesizingsecond chambers encapsulating the identified chambers. In some suchembodiments, the original chambers having degradable polymer matrix wallmay be degraded by treatment with a degradation agent which leaves thesecond chambers intact, which permits the biological components ofinterest to be isolated.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, where only illustrative embodiments of the presentdisclosure are shown and described. As will be realized, the presentdisclosure is capable of other and different embodiments, and itsseveral details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows a schematic illustration of a portion of a channel disposedin a fluidic device, according to some embodiments.

FIG. 2A shows a portion of a system as provided herein including anenergy source, according to some embodiments.

FIG. 2B shows a polymer matrix being formed around a biologicalcomponent in a portion of a system as provided herein, according to someembodiments.

FIG. 2C shows a method of forming a polymer matrix around a biologicalcomponent in a system as provided herein, according to some embodiments.

FIG. 3 is a flow chart depicting an embodiment of forming a polymermatrix.

FIG. 4A shows a portion of a channel including capture elements in afluidic device, according to some embodiments.

FIG. 4B illustrates biological components coupled to capture elements ona surface of a portion of a channel in a fluidic device, according tosome embodiments.

FIG. 4C illustrates polymer matrices disposed around biologicalcomponents in a portion of a channel of a fluidic device, according tosome embodiments.

FIG. 5 is a flow chart depicting an embodiment of forming a polymermatrix around a biological component coupled to a surface.

FIG. 6A shows a portion of another embodiment of a fluidic deviceincluding a sealable aperture.

FIG. 6B shows a method of trapping biological components in a portion ofa fluidic device, according to some embodiments.

FIG. 7A shows a top view of a schematic of a portion of a fluidicdevice, according to some embodiments.

FIG. 7B shows a top view of a schematic of a portion of a fluidic deviceincluding polymer matrices, according to some embodiments

FIG. 8 shows a top view of a schematic of a portion of a fluidic deviceincluding multiple different reagents, according to some embodiments.

FIG. 9A shows a portion of a spatial energy modulating element and acylindrical polymer matrix, according to some embodiments.

FIG. 9B shows a portion of a spatial energy modulating element andpolymer matrices in the shape of hollow cylinders, according to someembodiments.

FIG. 10 shows a micrograph of polymer matrix compartments encapsulatingone or more biological components, according to some embodiments.

FIG. 11A illustrates open compartments formed in a multi-step polymermatrix formation process, according to some embodiments.

FIG. 11B illustrates closed compartments formed in a multi-step polymermatrix formation process, according to some embodiments.

FIG. 12A is a schematic illustration of a portion of a surface of afluidic device coated with repelling elements, according to someembodiments.

FIG. 12B shows a micrograph of biological components captured on asurface using repelling elements, according to some embodiments.

FIG. 12C shows a higher magnification micrograph of biologicalcomponents captured on a surface using repelling elements, according tosome embodiments.

FIG. 13A is a schematic illustration of cell capturing and cell lysissteps of an exemplary mRNA 3′ gene expression workflow with externalsequencing.

FIG. 13B is a schematic illustration of generating a cDNA library from atemplate mRNA in an exemplary mRNA 3′ gene expression workflow withexternal sequencing.

FIG. 13C is a schematic illustration of a sequence library prep in anexemplary mRNA 3′ gene expression workflow with external sequencing.

FIGS. 14A and 14B show schematic illustrations of an exemplary mRNA 3′gene expression workflow with in-situ sequencing.

FIG. 15 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 16A illustrates an example of forming a polymer matrix using anenergy modulating element, according to some embodiments.

FIG. 16B illustrates a top view of an example of a polymer matrix formedusing an energy modulating element, according to some embodiments.

FIGS. 17A-17E illustrate exemplary steps of forming a polymer matrixusing a movable energy modulating element, according to someembodiments.

FIG. 18 shows a schematic of a whole-genome workflow, according to someembodiments.

FIG. 19 shows a schematic of a multi-omics workflow, according to someembodiments.

FIG. 20 shows polymer matrices inside a well plate format, according tosome embodiments.

FIG. 21 shows polymer matrices inside 10 mm size wells, according tosome embodiments.

FIG. 22 shows a fluidic device with a porous membrane on top of thepolymer matrices, according to some embodiments.

FIG. 23 shows a well plate with hydrogel traps inside the wells and aporous membrane on top of the polymer matrices, according to someembodiments.

FIG. 24A shows a microscope equipped with a DMD and integrated UVillumination LED, according to some embodiments.

FIG. 24B shows a schematic illustration of a DMD projecting a virtualmask image onto a fluidic channel filled with polymer precursor togenerate a polymer matrix, according to some embodiments.

FIG. 24C shows various virtual mask images that were projected using aDMD and the corresponding polymer matrices generated inside a fluidicdevice, according to some embodiments.

FIG. 25A shows a hydrogel structure in which individual cells areencapsulated in circular hydrogel matrices, according to someembodiments.

FIG. 25B shows cells identified using bright field imaging using 4×microscope objective, the resulting Voronoi mask calculated based on thelocation of the cells, and the hydrogel structures surrounding the cellsgenerated using the Voronoi mask, according to some embodiments.

FIG. 26A illustrates selective retention of fluorescent cells in amixture of fluorescent calcein AM stained cells and non-fluorescentcells, according to some embodiments.

FIG. 26B illustrates selective retention of cells of interest andremoval of cells that are not desired, according to some embodiments.

FIG. 27A shows hydrogel matrices inside a fluidic channel with a singlecell inside each chamber, or CellCage™ structure, according to someembodiments.

FIG. 27B is a spatial plot showing location of all the DNA clusters withsequences aligning to mRNA molecules from Jurkat cells, according tosome embodiments.

FIG. 27C shows a highlighted hydrogel matrix with a cell inside using ahigher magnification image and a corresponding spatial plot of DNAsequences mapping to human mRNA, according to some embodiments.

FIG. 27D shows a highlighted hydrogel matrix with a cell inside using ahigher magnification image and a corresponding spatial plot of DNAsequences mapping to human mRNA, according to some embodiments.

FIG. 28A is a scatterplot showing the number of reads uniquely mappingto the mouse and human genome within each hydrogel matrix, according tosome embodiments.

FIG. 28B shows spatial plots of DNA sequences mapping to human and mousemRNA, according to some embodiments.

FIG. 29A shows antibody labels flanked with an oligo sequence, abarcode, and a polyA tail, according to some embodiments.

FIG. 29B shows the relative expression of various surface proteinsdetected after sequencing antibody barcode oligos captured after lysingcells inside a hydrogel matrix, according to some embodiments.

FIG. 29C shows representative cells inside cell cages and spatial plotsof barcode sequence, color coded by the identity of the barcodesequence, illustrating abundance of various antibodies inside eachhydrogel matrix after cell lysis, according to some embodiments.

FIG. 30A shows the fluorescent signals given off from streptavidin beadsthat have captured IgG molecules secreted from CHO DP-12 cells,according to some embodiments.

FIG. 30B shows the fluorescent signal given off from streptavidin beadsthat have captured IgG molecules secreted from one CHO DP-12 cellcaptured in a hydrogel matrix, according to some embodiments.

FIG. 30C shows a spatial plot of RNA sequences mapping to the CHO DP-12cell captured in the hydrogel matrix of FIG. 30B, according to someembodiments.

FIG. 31A shows a schematic illustration of a microfluidic devicecontaining cytokine capture beads and mRNA capture oligos, according tosome embodiments.

FIG. 31B shows the gene expression analysis of non-stimulated &stimulated Jurkat cells, according to some embodiments.

FIG. 31C shows the fluorescent signal from two hydrogel matrices, eachcontaining a single IL-2 secreting cell, according to some embodiments.

FIG. 32 shows images of a hydrogel matrix being used to culture a CHOcell at 0 hours, 18 hours, 42 hours, and 46 hours, according to someembodiments.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION Introduction

Analyzing biological samples on a level of a cell's physical properties,proteome, transcriptome, genome, epigenome, methylome, secretome, ormetabolome can be performed on single cells or on a population of cells.For example, genetic material from a cell can be analyzed separatelyfrom, or in combination with, genetic materials from other cells. Whileanalyzing components of a sample individually may generate data withhigher resolution and less noise or cross-contamination, this approachcan be costly and time consuming. On the other hand, analyzing a sampleor a plurality of samples in bulk can be cost and time efficient but maygenerate undesired, or less desired, output (e.g., due toheterogeneity). For example, a particular datum may not be trackable toa source in the sample. A spatial association of the sample componentsmay also be lost. For example, two components of a sample may beadjacent to one another in their biologically relevant state but afterpooling a plurality of samples or components of a sample, the spatialinformation between the two components may be lost.

To avoid generating heterogenous samples, individual components of thesample may be compartmentalized. This can allow individual components ofa sample to be processed simultaneously, or substantiallysimultaneously, while retaining spatial information intact and reducingprocessing steps. Additionally, by preventing cross-contamination andloss of material during extraneous sample processing, the generated datamay be of higher quality (e.g., the data may have a higher signal tonoise ratio as appropriate). Some methods may preserve spatialinformation by barcoding individual or groups of compartments in asample. These methods may combine the barcoded components and may notperform multiple assays on the same component within a compartment oranalysis chamber. Some of these methods may require nucleotideamplification steps that can introduce biases in the generated data.

In order to compartmentalize individual components of a biologicalsample, a polymer matrix (e.g., a hydrogel matrix) can be formedadjacent to or around at least of portion of an individual component ina fluidic device. The hydrogel matrix may be selectively generated tosurround a component after the system detects the component or hydrogelmatrices can be generated according to a predefined pattern in a fluidicdevice. The hydrogel matrix may allow reagents and smaller entities topass while retaining the individual component of the biological samplein place. Because one or more individual components can be localizedwithin a fluidic device (e.g., encapsulated) and the localizedcomponents be exposed to one or more reagents and/or washing solutionsduring and/or in between analyses, multiple assays can be performedwithin the compartments (e.g., simultaneously, substantiallysimultaneously, serially, etc.).

Different assays may be performed in different locations of the fluidicdevice, for example, to test effects of different treatment conditions.Additionally, because components are not generally mixed and combined,low concentrations of components (e.g., due to dilution) can beprevented. For example, when analyzing genomic material, anamplification step can be avoided due to the preservation of the geneticmaterial in each compartment. By having two or more components within acompartment, interactions between components can be studied as well. Thepolymer matrix can be degradable “on demand” allowing for controlledlocalization and release mechanisms. The solutions provided herein canretain spatial information of the components and generate data on acellular, proteomic, transcriptomic, or genomic level. Since spatialinformation is retained, the data can be associated (e.g., linked) withphenotypic data. Further, the solutions provided herein can retainspatial information of the components and link data (e.g., phenotypicdata) on a cellular, proteomic, transcriptomic, or genomic level.

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Whenever the term “at least” precedes the first numerical value in aseries of two or more numerical values, the term “at least” applies toeach of the numerical values in that series of numerical values. Forexample, at least 1, 2, or 3 is equivalent to at least 1, at least 2, orat least 3.

Whenever the term “less than” precedes the first numerical value in aseries of two or more numerical values, the term “less than” applies toeach of the numerical values in that series of numerical values. Forexample, less than 3, 2, or 1 is equivalent to less than 3, less than 2,or less than 1.

The terms “coupled to,” “connected to,” and “in communication with,” asused herein, generally refer to any form of interaction between two ormore entities, including mechanical, electrical, magnetic,electromagnetic, fluid, biological, and thermal interaction. Twocomponents may be coupled to each other even though they are not indirect contact with each other.

The terms “polypeptide” and “peptide,” as used interchangeably herein,generally refer to a polymer of amino acids in which an amino acid maybe linked to another amino acid by a peptide bond. In some examples, apolypeptide is a protein. The amino acid may be a naturally occurringamino acid or a non-naturally occurring amino acid (e.g., an amino acidanalogue). The polypeptide can be linear or branched. The polypeptidecan include modified amino acids. The polypeptide may be interrupted bynon-amino acids. A polypeptide can occur as a single chain or anassociated chain. The polypeptide may include a plurality of aminoacids. The polypeptide may have a secondary and tertiary structure(e.g., the polypeptide may be a protein). In some examples, thepolypeptide can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 100, 1,000, 10,000, or more amino acids. The polypeptide may bea fragment of a larger polymer. In some examples, the polypeptide can bea fragment of a larger polypeptide, such as a fragment of a protein.

The term “amino acid,” as used herein, generally refers to a naturallyoccurring or non-naturally occurring amino acid (e.g., an amino acidanalogue). The non-naturally occurring amino acid may be an engineeredor synthesized amino acid.

The term “sample,” as used herein, generally refers to a chemical orbiological sample containing a biological component. The biologicalcomponent may comprise a cell, a nucleic acid, a microbiome, a protein,a combination of cells, a metabolite, a combination thereof, or anyother suitable component of a biological sample. For example, a samplecan be a biological sample including one or more cells. For anotherexample, a sample can be a biological sample including one or morepolypeptides. The biological sample can be obtained (e.g., extracted orisolated) from or include blood (e.g., whole blood), plasma, serum,urine, saliva, mucosal excretions, sputum, stool, and tears. Thebiological sample can be a fluid or tissue sample (e.g., skin sample).In some instances, the sample may be derived from a homogenized tissuesample (e.g., brain homogenate, liver homogenate, or kidney homogenate).In certain embodiments, the sample may include a specific type of cell(e.g., a neuronal cell, muscle cell, liver cell, or kidney cell,). Thesample may comprise or be acquired from a diseased cell or tissue (e.g.,a tumor cell or a necrotic cell), In some embodiments, the sample mayinclude or may be from a disease-associated inclusion (e.g., a plaque, abiofilm, a tumor, or a non-cancerous growth). In certain embodiments,the sample may include or may be obtained from a cell-free bodily fluid,such as whole blood, saliva, or urine. In various embodiments, thesample can include circulating tumor cells. In some cases, the samplemay include or may be an environmental sample (e.g., soil, waste, orambient air), industrial sample (e.g., samples from any industrialprocesses), or a food sample (e.g., dairy product, vegetable product, ormeat product). The sample may be processed prior to loading into amicrofluidic device. For example, the sample may be processed to purifya certain cell type or polypeptide and/or to include reagents.

As used herein, the term “polymer matrix” generally refers to a phasematerial (e.g., continuous phase material) that comprises at least onepolymer. In some embodiments, the polymer matrix refers to the at leastone polymer as well as the interstitial space not occupied by thepolymer. A polymer matrix may be composed of one or more types ofpolymers. A polymer matrix may include linear, branched, and crosslinkedpolymer units. A polymer matrix may also contain non-polymeric speciesintercalated within its interstitial spaces not occupied by polymerchains. The intercalated species may be solid, liquid, or gaseousspecies. For example, the term “polymer matrix” may encompass desiccatedhydrogels, hydrated hydrogels, and hydrogels containing glass fibers. Apolymer matrix may comprise a polymer precursor, which generally refersto one or more molecules that upon activation can trigger or initiate apolymeric reaction. A polymer precursor can be activated byelectrochemical energy, photochemical energy, a photon, magnetic energy,or any other suitable energy. As used herein, the term “polymerprecursor” includes monomers (that are polymerized to produce a polymermatrix) and crosslinking compounds, which may include photo-initiators,other compounds necessary or useful for generating polymer matrices, andthe like.

As used herein, the term “physical photomask” generally refers to aphysical structure having a plurality of apertures or holes throughwhich light may be projected. Physical photomasks can be used to createhydrogel matrices as described herein by causing the polymer precursorsolution to polymerize and forming three-dimensional structures thatcorrespond to the pattern on the photomask. A physical photomask can bepatterned with a specific layout or geometric pattern. A physicalphotomask may be adhered to the upper surface of a flow cell.

In some embodiments, as used herein, the term “local parameter” means avalue of a parameter (such as, pH) in or immediately adjacent to achamber formed by polymer matrix walls.

As used herein, the term “on demand” means an operation may be directedto individual, discrete, selected locations (e.g. a spatial location ofpolymer precursor solution; or a selected polymer matrix chamber). Suchselection may be based on manual observation of optical signals or datacollected by a detector, or such selection may be based on a computeralgorithm operating on optical signals or data collected by a detector.Manual observation of optical signals or data collected by a detectorcan include either real-time detection or detection at a time periodprior to modulating a unit of energy to polymerize polymer precursors ordegrading a chamber. For example, a subset of chambers (all formed withphoto-degradable polymer matrix walls) may be pre-selected for releasingand removing their contents based on position information and the valuesof optical signals from an analytical assay carried out in the chambers.The pre-selected chambers may be photo-degraded by selectivelyprojecting a light beam of appropriate wavelength characteristics (forexample, with the spatial energy modulating element) to degrade thepolymer matrix walls of the pre-selected chambers. In another example, aplurality of chambers may be observed in real-time (e.g. via fluorescentmicroscopy) for detection of an analyte of interest and one or morechambers of the plurality of chambers is selected, in real-time, upondetection of the analyte of interest, for degradation.

As used herein, the terms “microfluidic” and “nanofluidic” in referenceto devices are used interchangeably herein, each means an integratedsystem for capturing, moving, mixing, dispensing or analyzing smallvolumes of fluid, including samples (which, in turn, may contain orcomprise cellular or molecular analytes of interest), reagents,dilutants, buffers, or the like. Generally, reference to “microfluidics”and “nanofluidics” denotes different scales in the size of devices andvolumes of fluids handled. In some embodiments, features of amicrofluidic device have cross-sectional dimensions of less than a fewhundred square micrometers and have passages, or channels, withcapillary dimensions, for example, having maximal cross-sectionaldimensions of from about 500 μm to about 0.1 μm. In some embodiments,microfluidics devices have volume capacities in the range of from 1 μLto a few nL, e.g. 10-100 nL. Dimensions of corresponding features, orstructures, in nanofluidics devices are typically from 1 to 3 orders ofmagnitude less than those for microfluidics devices. One skilled in theart would know from the circumstances of a particular application whichdimensionality would be pertinent. In some embodiments, microfluidic ornanofluidic devices have one or more chambers, ports, and channels thatare interconnected and in fluid communication and that are designed forcarrying out one or more analytical reactions or processes, either aloneor in cooperation with an appliance or instrument that provides supportfunctions, such as sample introduction, fluid and/or reagent drivingmeans, such as positive or negative pressure, acoustical energy, or thelike, temperature control, detection systems, data collection and/orintegration systems, and the like. In some embodiments, microfluidicsand nanofluidics devices may further include valves, pumps, filters andspecialized functional coatings on interior walls, e.g. to preventadsorption of sample components or reactants, facilitate reagentmovement by electroosmosis, or the like. Such devices may be fabricatedas an integrated device in a solid substrate, which may be glass,plastic, or other solid polymeric materials, and may have a planarformat for ease of detecting and monitoring sample and reagent movement,especially via optical or electrochemical methods. In some embodiments,such devices are disposable after a single use. The fabrication andoperation of microfluidics and nanofluidics devices are well-known inthe art as exemplified by the following references that are incorporatedby reference: Ramsey, U.S. Pat. Nos. 6,001,229; 5,858,195; 6,010,607;and U.S. Pat. No. 6,033,546; Soane et al, U.S. Pat. Nos. 5,126,022 and6,054,034; Nelson et al, U.S. Pat. No. 6,613,525; Maher et al, U.S. Pat.No. 6,399,952; Ricco et al, International patent publication WO02/24322; Bjornson et al, International patent publication WO 99/19717;Wilding et al, U.S. Pat. Nos. 5,587,128; 5,498,392; Sia et al,Electrophoresis, 24: 3563-3576 (2003); Unger et al, Science, 288:113-116 (2000); Enzelberger et al, U.S. Pat. No. 6,960,437; Cao,“Nanostructures & Nanomaterials: Synthesis, Properties & Applications,”(Imperial College Press, London, 2004); Haeberle et al, LabChip, 7:1094-1110 (2007); Cheng et al, Biochip Technology (CRC Press, 2001); andthe like.

As used herein, the term “analyte” generally refers to a discretebiological or chemical entity to be measured, detected, and/ordistinguished using the methods and systems described herein. In someembodiments, an analyte may be a biological component as describedherein.

Systems for Analysis of Biological Components

The present disclosure provides systems for compartmentalizing orisolating one or more biological components. The system can include afluidic device containing or including one or more biologicalcomponents. The fluidic device may contain or include one or morepolymer precursors. In some cases, the fluidic device can comprise afirst surface configured to couple or receive at least one of the one ormore biological components to form a coupled biological component. Thesystems may also include at least one energy source, wherein the energysource is in communication with the fluidic device. In some embodiments,the energy source may be in optical communication with the fluidicdevice. In various embodiments, the at least one energy source may forma polymer matrix on or adjacent to at least a portion of the one or morebiological components.

In some cases, a sample may be introduced of provided to the system. Incertain cases, the sample may comprise one or more biologicalcomponents. The system may be used to separate one or more biologicalcomponents from one another. In various cases, the biological componentsmay be physically separated. In some cases, the biological componentsmay be in fluidic communication with one another. In certain cases, thebiological components may be in chemical communication with one another.The system may be used for single-cell analysis. In some embodiments,the system may be used for single-cell analysis on a genome level. Forexample, the system may be used for genome sequencing. For anotherexample, the system may be used for deoxyribonucleic acid (DNA)sequencing. The system may be used for DNA sequencing of cell-free DNA,whole genome sequencing, whole exome sequencing, targeted sequencing, or16S sequencing. The system may be used for studying DNA tags attached tobiomolecules of interest. The biomolecules may comprise proteins,metabolites, etc. In some cases, the DNA may be a nuclear DNA or amitochondrial DNA. The system may be used for single-cell or bulkanalysis on a transcriptome level. For example, the system may be usedfor ribonucleic acid (RNA) sequencing. For example, the system may beused for 3′ or 5′ gene expression analysis, immune repertoire study of acell, or full-length mRNA analysis. In some embodiments, the system maybe used for single-cell analysis on a proteome level. The system may beused for functional assay(s) of a biological component. The system maybe used for studying surface proteins, secreted proteins, or metabolitesof a biological component. In some cases, the system may be used tomeasure a quality of a biological component. In some cases, the measuredquality may be the size or shape of a biological component. In somecases, the system may be used to study epigenomics, DNA methylation, orchromatin accessibility in a biological component. The system may beused for other suitable assays, experiments, and processes.

In certain embodiments, the system may be used for single-cell analysison an indirect cell-cell interaction level. For example, an effect ofone or more molecules produced from a first cell on a second cell can beanalyzed using the system as provided herein. In various embodiments,the system may be used for analyzing direct cell-cell interactions. Forexample, two or more cells (e.g., a first cell and a second cell) can bein physical contact and the effect or effects of the first cell on thesecond cell, or vice versa, can be analyzed using the system asdisclosed herein. In some embodiments, the system may be used for drugresponse analysis in a biological component. In certain embodiments, thesystem may be used for analyzing a biological component's response tovarious physiological conditions (e.g., various media, temperature,mechanical stimuli, etc.).

In some cases, the sample comprises a biological sample. The biologicalsample may comprise a biological component. In some embodiments, thebiological sample may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10²⁰, or more biological components. The biologicalsample may include any number of biological components between any ofthe two numbers mentioned herein. In some embodiments, the biologicalsample may comprise more than 10²⁰ biological components. The biologicalcomponent may comprise a cell. In some embodiments, the cell maycomprise a eukaryotic cell, a prokaryotic cell, a fungal cell, aprotozoan, an algal cell, a plant cell, an animal cell (e.g., a humancell), or any other suitable cell. The biological component may comprisea cell, a virus, a bacterium, a nucleic acid (e.g., DNA, or RNA), aprotein, or a combination thereof. The combination may comprise aDNA-protein complex, an RNA-protein complex, or a combination thereof.In certain embodiments, a nucleic acid may comprise DNA. The DNA may beat least 10 base pair (bp) long. In some embodiments, the DNA is atleast 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp long, orlonger than 800 bp.

Polymer precursors may be formed by using any hydrogel precursor andcrosslinkers of Table 1A (columns 1 and 3, respectively). The resultingpolymer matrices may be degraded with the indicated degradation agentsin Table 1A (column 4).

TABLE 1A Precursors Hydrogels Crosslinkers Degradation Agents AcrylamidePolyacrylamide Bis-acryloyl cystamine (structure 1) DTT/TCEP/THPPEG-based PEG Bis(2-methacryloly)oxyethyl disulfide DTT/TCEP/THPacryloyl (structure 2) Dextran-based DextranN,N′-(1,2-Dihydroxylethylene)bis- NaIO4 acryloyl acrylamide structure(3) Polysaccharide- Polysaccharide Structure 4 NaOH, ethanolamine baseacryloyl DTT/TCEP/THP Gelatin-base Gelatin Structure 5 NaOH,ethanolamine, acryloyl nucleophilic bases Structure 6 NaOH, alkali,organic bases Structure 7 Acid

TABLE 1B Structure Number Structure 1

2

3

4

5

6

7

In certain embodiments, one or more polymer precursors may be added toor included with the biological sample. One or more biological samplesand one or more polymer precursors may be introduced into the system(e.g., into the fluidic device of the system). The one or morebiological samples and the one or more polymer precursors may beintroduced into the fluidic device in any order (e.g., in parallel,sequentially, etc.). For example, the biological sample(s) may beintroduced prior to the polymer precursor(s), the polymer precursor(s)may be introduced prior to the biological sample(s), the biologicalsample(s) and polymer precursor(s) may be introduced simultaneously (orsubstantially simultaneously), or in any other suitable manner or order.In some embodiments, a polymer precursor may include one or morehydrogel precursors. The one or more polymer precursors may be storedand/or introduced separately into the system. In some cases, the one ormore polymer precursors may be mixed with the one or more biologicalcomponents prior to introduction into the system. In various cases, theone or more polymer precursors may be mixed with the one or morebiological components after introduction into the system.

The system may comprise a fluidic device. In some embodiments, thefluidic device may include one or more polymer precursors. In otherwords, one or more polymer precursors may be disposed within at least aportion of the fluidic device (e.g., within at least a portion of achannel of the fluidic device). In some embodiments, the fluidic devicemay comprise one or more channels or chambers. In some embodiments, thefluidic device may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000channels or chambers, or any number of channels or chambers between anyof the two numbers mentioned herein. In some embodiments, the fluidicdevice comprises more than 10,000 channels or chambers. As describedherein, the fluidic device may include one or more channels. The fluidicdevice may also, or alternatively, include one or more chambers. Theterms channel and chamber may be used interchangeably in the disclosureherein unless indicated otherwise. For example, a channel or a chamberof the fluidic device may comprise a first surface, a second surface, ormore surfaces.

A channel or chamber of a fluidic device (also sometimes referred to asa “flow chamber,” or “reaction chamber,” as opposed to a chamber that isformed from polymer matrix walls within a channel) may receive or beconfigured to receive a biological sample. FIG. 1 shows a schematicillustration of a portion of a channel 100 that may be disposed in atleast a portion of a fluidic device of a system as provided herein. Thefluidic device may comprise a channel 100. The channel 100 may comprisea first surface 101. Further, the channel 100 may comprise a secondsurface 102. In some embodiments, the first surface 101 and the secondsurface 102 are disposed, placed, or positioned opposite of one another(e.g., as depicted in FIG. 1). In some embodiments, the first surfaceand second surface are substantially parallel, so that the perpendiculardistance between them is substantially the same throughout the channel,for example, where chambers are formed. In some embodiments, theperpendicular distance between a first surface and a second surfacedepends in part and the nature and size of the biological components tobe analyzed. In some embodiments, such as, those adapted to analyzingmammalian cells, the perpendicular distance between a first surface anda second surface may be in the range of from 10 μm to 500 μm, or in therange of from 50 μm to 250 μm. In some embodiments, the perpendiculardistance between a first surface and a second surface may be in therange of from twice the average size of the biological component to beanalyzed to five times the average size of the biological component tobe analyzed. In some embodiments, the perpendicular distance between afirst surface and a second surface may be in the range of from twice theaverage size of the largest biological component in the biologicalsample to five times the average size of the largest biologicalcomponent in the biological sample. In some embodiments, the firstsurface 101 may be a lower surface. In certain embodiments, the secondsurface 102 may be an upper surface. The terms “lower” and “upper” arenot intended to be limiting and are used herein for convenience whenreferring to the figures. The channel 100 may receive a biologicalsample comprising one or more biological components 50, 51. The channel100 may receive one or more polymer precursors. As illustrated in FIG.1, the biological components 50, 51 may include cells. However, asdiscussed herein, the biological components may include tissues,proteins, nucleic acids, etc. In some embodiments, the first surface101, the second surface 102, or both surfaces may couple or receive, orbe configured to couple or receive, at least one of the one or morebiological components 50, 51. In some cases, the first surface 101 maycouple or receive, or be configured to couple or receive, a biologicalcomponent (e.g., biological components 50, 51). In certain cases, thesecond surface 102 may couple or receive, or be configured to couple orreceive, a biological component (e.g., biological components 50, 51).

In certain cases, a channel may have a rectangular, circular,semi-circular, oval cross-section, or other suitably shapedcross-section. Accordingly, the channel may have a single, internalsurface. In some cases, a channel may have a triangular, square,rectangular, polygonal, or other cross-section. Accordingly, the channelmay have three or more internal surfaces. One or more of the internalsurfaces may be couple or receive, or be configured to couple orreceive, the one or more biological components.

In some cases, the first surface 101, the second surface 102, or bothsurfaces 101, 102 may be functionalized, for example, with a coating(e.g., a surface coating). In some embodiments, the surface coating maybe a surface polymer. Some non-limiting examples of surface coatings mayinclude a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), afunctional group to capture one or more moieties (e.g., a chemicalmoiety), an acrylamide, an agarose, a biotin, a streptavidin, astrep-tag II, a linker, a functional group comprising an aldehyde, aphosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide,an aldehyde dithiolane, or a combination thereof. In variousembodiments, the surface coating may include a functional group tocapture one or more moieties. For example, the acrylamide, the agarose,etc. may include such a functional group. In certain embodiments, thesurface polymer may comprise polyethylene glycol (PEG), a thiol, analkene, an alkyne, an azide, or combinations thereof. In variousembodiments, the surface polymer may comprise a silane polymer. In someembodiments, the surface polymer may be functionalized with at least oneof an oligonucleotide, an antibody, a cytokine, a chemokine, a protein,an antibody derivative, an antibody fragment, a carbohydrate, a toxin,or an aptamer.

In some cases, the first surface 101, the second surface 102, or bothsurfaces 101, 102 may comprise one or more barcodes (e.g., nucleic acidbarcodes). In some embodiments, the first surface 101, the secondsurface 102, or both surfaces 101, 102 may comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000,10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000,5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodesbetween any of the two numbers mentioned herein. The barcodes may coveran area of about 500 nm² to about 500 μm². In some embodiments, thefirst surface 101, the second surface 102, or both surfaces 101, 102 maycomprise at most about 10,000,000 total number of barcodes. The barcodesmay be different from one another (e.g., each barcode may be unique). Incertain embodiments, a first portion or subset of the barcodes may bedifferent from a second portion or subset of the barcodes. There may be2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions orsubsets of the barcodes, or any number of portions or subsets of thebarcodes between any of the two numbers mentioned herein. In some cases,a barcode (or a portion/subset of barcodes) may be associated with thelocation of the barcode on a surface (location coordinates (e.g., x-,y-coordinates) on a surface of a channel). A barcode may be attached toor coupled to the captured biological component. In some embodiments,the barcode may be a unique identifier that distinguishes a biologicalcomponent from other biological components (e.g., that identifies afirst biological component versus a second biological component). Insome embodiments, a barcode may comprise a nucleic acid sequence (e.g.,common sequence) to capture a biological component, or used inamplification. In some embodiments, a barcode may comprise a uniqueidentifier comprising a unique nucleic acid sequence (e.g., DNAsequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. Insome embodiments the barcode may comprise a fluorescent molecule. Insome embodiments, a location of the captured biological component may beassociated with the unique identifier to, for example, retain spatialinformation of a biological component.

In some embodiments, the fluidic device may be a flow cell. For example,the fluidic device may be used for sequencing (e.g., DNA or RNAsequencing). In some embodiments, the fluidic device may be amicrofluidic device. In certain embodiments, the fluidic device may be ananofluidic device.

The system disclosed herein may comprise one or more energy sources. Theenergy source may be in communication with the fluidic device. In someembodiments, the energy source may be in optical communication with thefluidic device. In some cases, the energy source can be used to form oneor more polymer matrices in the fluidic device (e.g., on or adjacent toa surface of a channel or chamber of the fluidic device). In someembodiments, the energy source may comprise a light generating device, aheat generating device, an electrochemical reaction generating device,an electrode, or a microwave device. A polymer matrix may be formed in achannel of the fluidic device. The energy source may direct or transferenergy to a predetermined position in the fluidic device. The energy maycause or activate the one or more polymer precursors to form a polymermatrix (e.g., to polymerize) in the predetermined position.

In some embodiments, the polymer matrix may comprise a hydrogel. In someembodiments, the hydrogel may be porous enough, or have pores of asuitable size, to allow movement or transfer of a reagent (e.g., anenzyme, a chemical compound, a small molecule, an antibody, etc.)through the polymer matrix, while the hydrogel may not allow movement ortransfer of the biological component (e.g., DNA, RNA, a protein, a cell,etc.) through the polymer matrix. In some embodiments, the pores mayhave a diameter from 5 nm to 100 nm. In some embodiments, the pores mayhave a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameterlarger than 100 nm. In some embodiments, the pores may have a diametersmaller than 5 nm. The reagent may comprise an enzyme or a primer havinga size of less than 50 base pairs (bp). A primer may comprise asingle-stranded DNA (ssDNA). In some embodiments, a primer may have asize from 5 bp to 50 bp. In some embodiments, a primer may have a sizefrom 5 bp to 10 bp, 10 bp to 20 bp, from 20 bp to 30 bp, 30 bp to 40 bp,or 40 bp to 50 bp. In some embodiments, a primer may have a size of morethan 50 bp. In certain cases, a primer may have a size of less than 5bp. A reagent may comprise a lysozyme, a proteinase K, hexamers (e.g.,random hexamers), a polymerase, a transposase, a ligase, a catalyzingenzyme, a deoxyribonuclease, a deoxyribonuclease inhibitor, aribonuclease, a ribonuclease inhibitor, DNA oligos, deoxynucleotidetriphosphates, buffers, detergents, salts, divalent cations, or anyother suitable reagent.

In some embodiments, the one or more reagents flow through a membrane.In some embodiments, the membrane is semi-permeable. In someembodiments, the membrane comprises pores. In some embodiments, thepores are less than 1 micrometer wide. In some embodiments, the width ofthe pores is about 0.5 micrometers to about 15 micrometers. In someembodiments, the width of the pores is about 0.5 micrometers to about 1micrometer, about 0.5 micrometers to about 5 micrometers, about 0.5micrometers to about 10 micrometers, about 0.5 micrometers to about 15micrometers, about 1 micrometer to about 5 micrometers, about 1micrometer to about 10 micrometers, about 1 micrometer to about 15micrometers, about 5 micrometers to about 10 micrometers, about 5micrometers to about 15 micrometers, or about 10 micrometers to about 15micrometers. In some embodiments, the width of the pores is about 0.5micrometers, about 1 micrometer, about 5 micrometers, about 10micrometers, or about 15 micrometers. In some embodiments, the width ofthe pores is at least about 0.5 micrometers, about 1 micrometer, about 5micrometers, or about 10 micrometers. In some embodiments, the width ofthe pores is at most about 1 micrometer, about 5 micrometers, about 10micrometers, or about 15 micrometers. In some embodiments, the one ormore reagents is an enzyme, a drug molecule, oligonucleotide, primer, orany combination thereof. In some embodiments, the one or more reagentsis a lysis reagent. In some embodiments, the one or more reagents is anucleic acid denaturation reagent. In some embodiments, the one or morereagents degrades the polymer matrix.

FIG. 22 shows a fluidic device with a porous membrane 2210 on top. Thebiological components 2202 are encapsulated and/or localized on thesurface of a fluidic device 2206 using polymer matrices 2204. Reagentsthat fit through the pores in the membrane 2210 can enter the polymermatrices and react with the biological component 2202. In someembodiments, as shown in FIG. 23, the polymer matrices 2304 andbiological components 2302 are seeded on a well plate 2306, and a porousmembrane 2310 sits on top of the polymer matrices 2304. Reagents thatfit through the pores in the membrane 2310 can enter the polymermatrices and react with the biological component 2302. Due to the wallsof the well 2308, there may be no fluidic communication betweendifferent wells, so different reagents can be used in each individualwell.

FIG. 2A shows a portion of a system as provided herein including anenergy source 203. The embodiment of FIG. 2A may include components thatresemble components of FIG. 1 in some respects. For example, theembodiment of FIG. 2A includes a channel 200 that may resemble thechannel 100 of FIG. 1. It will be appreciated that the illustratedembodiments may have analogous features. Accordingly, like features aredesignated with like reference numerals, with the leading digitsincremented to “2.” Relevant disclosure set forth above regardingsimilarly identified features thus may not be repeated hereafter.Moreover, specific features of the system provided herein, and relatedcomponents shown in FIG. 2A may not be shown or identified by areference numeral in the drawings or specifically discussed in thewritten description that follows. However, such features may clearly bethe same, or substantially the same, as features depicted in otherembodiments and/or described with respect to such embodiments.Accordingly, the relevant descriptions of such features apply equally tothe features of the system and related components of FIG. 2A. Anysuitable combination of the features, and variations of the same,described with respect to the system and components illustrated in FIG.1, can be employed with the system and components of FIG. 2A, and viceversa. This pattern of disclosure applies equally to further embodimentsdepicted in subsequent figures and described hereafter.

With continued reference to FIG. 2A, the channel 200 of the system mayinclude a first surface 201 and a second surface 202. In someembodiments, the energy source 203 may comprise one or more energyemitting portions (e.g., an energy emitting portion 205). In someembodiments, the energy source 203 may comprise one or more non-emittingportions (e.g., a non-emitting portion 204). The non-emitting portion204 may not emit, or be configured to emit, energy. In some embodiments,the emitting portion 205 can emit energy in the form of electromagneticwaves (e.g., microwaves, light, heat, etc.) to at least a portion of thefluidic device. In certain embodiments, the emitting portion 205 canemit energy to the fluidic device. In some embodiments, the fluidicchannel may be coupled to on a movable stage. In other embodiments,light may be projected to or onto at least a portion of the fluidicchannel to generate one or more polymer matrices. The light may bedirected to various parts of the fluidic channel. In some embodiments,the emitting portion 205 may be coupled to an objective (e.g., amicroscope objective or lens), where the objective may be moved todifferent portions of the fluidic device. The objective may provide ashape (e.g., virtual or physical mask) to allow light to form a patternon the fluidic device, in order to form a polymer matrix similar orcomplementary to the pattern. In various embodiments, the one or morepolymer precursors in the fluidic device or mixed with the biologicalsample can absorb emitted energy 206. In some embodiments, the emittedenergy 206 can form, or be sufficient to form, a polymer matrix from theone or more polymer precursors. For example, a portion of the one ormore polymer precursors within the channel 200 of the fluidic device maybe activated by the emitted energy and a polymerization reaction may beinitiated to form a polymer matrix.

In some embodiments, the energy source may emit energy to a largerportion of the fluidic channel or almost the entire surface of thefluidic channel. A physical mask may be used to block the energy emittedto one or more portions of the fluidic channel. The energy source (e.g.,light source) may be coupled to the fluidic device via an objective(e.g., a microscope objective or lens). The energy source may bedirected to a portion of the fluidic channel (e.g., via a movableobjective). In some cases, the light source, the objective, and/or thefluidic channel are movable to allow emission of energy to the fluidicchannel so as to generate a pattern on at least a portion of a surfaceof the fluidic device. The polymer matrix may be formed similarly orcomplementary to the pattern of energy emission.

A polymer precursor may comprise an activating molecule that can absorbthe emitted energy 206 to initiate polymerization of the one or morepolymer precursors in the fluidic device. Non-limiting examples of theactivating molecule may include a photocatalyst, a photoactivator, aphotoacid generator, or a photobase generator. In some embodiments, afirst polymer matrix 208 and/or a second polymer matrix 209 can beformed on or adjacent to a biological component 50. In certainembodiments, the first polymer matrix 208 and the second polymer matrix209 can form an analysis chamber or compartment 220 that separates(e.g., physically separates) the biological component 50 from otherbiological components (e.g., biological components 51, 52, or 53) in thefluidic device. Stated another way, the polymer matrix maycompartmentalize the channel (e.g., channel 200). In variousembodiments, the polymer matrix may partially surround a biologicalcomponent. For example, a polymer structure surrounding a biologicalcomponent may form a closed structure (e.g., a hollow cylinder-shapedpolymeric structure) or a partially open structure (e.g., acrescent-shaped polymeric structure). In some embodiments, two or morepolymer matrices may be formed adjacent to a biological componentforming a compartment separating the biological component from otherbiological components. In certain embodiments, the polymer matrix maycomprise or form a wall (e.g., a polymer matrix wall).

In various embodiments, the polymer matrix comprises a hydrogel. In someembodiments, the polymer matrix wall may be a hydrogel wall. In someembodiments, a hydrogel or hydrogel wall may comprise polyethyleneglycol (PEG)-thiol, PEG-acrylate, acrylamide,N,N′-bis(acryloyl)cystamine (BAC), PEG, polypropylene oxide (PPO),polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methylmethacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lacticacid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone(PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid),poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin,alginate sulfate, dextran sulfate, dextran-acrylamide, hyaluronan,pectin, carrageenan, gelatin, chitosan, cellulose, collagen,bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone,diethylene glycol diallyl ether, ethylene glycol diacrylate,polymethyleneglycol diacrylate, poly(ethyleneglycol) diacrylate,trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate,ethoxylated pentaerythritol tetraacrylate, or combinations or mixturesthereof. A hydrogel or hydrogel wall may comprise a degradablecross-linker (e.g., N,N′-Bis(acryloyl) cystamine, chitosan,poly(ε-caprolactone) diacrylate, polylactide diacrylate, polylactidedimethacrylate, poly(lactide-co-glycolide, polycaprolactone molecules,or other suitable degradable cross-linkers).

In some embodiments, the surface of the polymer matrix or hydrogel maybe functionalized by coupling a functional group to the polymer matrixor hydrogel. Some non-limiting examples of functional group may includea capture reagent (e.g., pyridinecarboxaldehyde (PCA)), an acrylamide,an agarose, a biotin, a streptavidin, a strep-tag II, a linker, afunctional group comprising an aldehyde, a phosphate, a silicate, anester, an acid, an amide, an aldehyde dithiolane, PEG, a thiol, analkene, an alkyne, an azide, or a combination thereof. In some cases,the functionalized polymer matrix may be used to capture biomoleculesinside a polymer matrix compartment formed adjacent to (e.g., around oron) the biological component. The biomolecule may be produced by thebiological component (e.g., secretome from a cell). The functionalizedsurface of the polymer matrix inside the compartment may be used tocapture reagents or molecules from outside the compartment. Thefunctionalized surface may increase surface area covered by a reagent, amolecular sensor, or any molecule of interest (e.g., an antibody).

In some embodiments, the compartment surrounding a biological componentmay comprise a polygon base. In various embodiments, the compartmentsurrounding a biological component may comprise a circular or oval base(see, e.g., the compartment 220 or the compartment 222 in FIG. 2C). Incertain embodiments, the polymer matrix wall of the compartment may havea thickness (e.g., a width) from 1 μm to 250 μm. The polymer matrix wallmay have a thickness from 1 μm to 250 μm. The polymer matrix wall orcompartment may have a thickness from 1 μm to 5 μm, 1 μm to 10 μm, 1 μmto 20 μm, 1 μm to 30 μm, 1 μm to 40 μm, 1 μm to 50 μm, 1 μm to 100 μm, 1μm to 150 μm, 1 μm to 250 μm, 5 μm to 10 μm, 5 μm to 20 μm, 5 μm to 30μm, 5 μm to 40 μm, 5 μm to 50 μm, 5 μm to 100 μm, 5 μm to 150 μm, 5 μmto 250 μm, 10 μm to 20 μm, 10 μm to 30 μm, 10 μm to 40 μm, 10 μm to 50μm, 10 μm to 100 μm, 10 μm to 150 μm, 10 μm to 250 μm, 20 μm to 30 μm,20 μm to 40 μm, 20 μm to 50 μm, 20 μm to 100 μm, 20 μm to 150 μm, 20 μmto 250 μm, 30 μm to 40 μm, 30 μm to 50 μm, 30 μm to 100 μm, 30 μm to 150μm, 30 μm to 250 μm, 40 μm to 50 μm, 40 μm to 100 μm, 40 μm, to 150 μm,40 μm to 250 μm, 50 μm to 100 μm, 50 μm to 150 μm, 50 μm to 250 μm, 100μm to 150 μm, 100 μm to 250 μm, or 150 μm to 250 μm. The polymer matrixwall or compartment may have a thickness of about 1 μm, about 5 μm,about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about100 μm, about 150 μm, or about 250 μm. The polymer matrix wall orcompartment may have a thickness of at least 1 μm, 5 μm, 10 μm, 20 μm,30 μm, 40 μm, 50 μm, 100 μm, 150 μm, 250 μm, or more. The polymer matrixwall or compartment may have a thickness of at most 5 μm, 10 μm, 20 μm,30 μm, 40 μm, 50 μm, 100 μm, 150 μm, or 250 μm. The polymer matrix wallor compartment may have a thickness of less than 1 μm.

With continued reference to FIG. 2A, the polymer matrix 208, 209, or atleast a portion of the polymer matrix 208, 209, may be coupled to thefirst surface 201, the second surface 202, or both surfaces 201, 202. Incertain embodiments, the polymer matrix, or at least a portion of thepolymer matrix, may be coupled to a third surface, a fourth surface, afifth surface, etc. as appropriate. In various embodiments, the polymermatrix 208, 209 may extend from the first surface 201 to the secondsurface 202 (e.g., through at least a portion of a lumen of the channel200 or a cavity of a chamber) such that the polymer matrix surrounds, orsubstantially surrounds, the biological component 50. In someembodiments, two or more biological components (e.g., biologicalcomponents 50, 51 of FIG. 2C) that are in close physical proximity maybe separated (e.g., by agitating or shaking the fluidic device). Thefluidic device may be agitated or shaken by physical movement, use of asonic pulse, changing a flow in the channel, or any other suitablemethod of agitation. A polymer matrix may then be formed that surrounds(or partially surrounds) the biological components that are separated.FIG. 2B shows polymer matrices 208, 209 formed surrounding thebiological component 50 after being separated from the biologicalcomponent 51. FIG. 2C shows a process, according to various embodiments,of separating the two biological components 50, 51, which are in closeproximity. That is, by agitating or shaking the fluidic device thebiological components 50, 51 can be separated. In some embodiments,separation of the biological components is achieved through fluidicpressure, flow pulsation, dielectrophoresis, optothermal flow, or somecombination thereof. In some cases, separation of the biologicalcomponents is achieved through acoustic vibration. FIG. 2C also shows apolymer matrix being formed to generate a compartment 222 surroundingthe biological component 50 after the separation of the biologicalcomponents 50, 51.

With continued reference to FIG. 2A, in some cases, the energy source203 can, or be configured to, form or produce one or more emittingportions 205 and one or more non-emitting portions 204. The systemsdisclosed herein may further include a spatial energy modulating elementto direct energy from the energy source to one or more targeted portionsof the fluidic device. For example, the spatial energy modulatingelement may be configured to selectively direct the energy from theenergy source to form a polymer matrix in a discrete area of the fluidicdevice. In some embodiments, the discrete area is chosen based on thelocation of a biological component. In some embodiments, the area of thediscrete area is less than the area of the fluidic device. In someembodiments, a biological component is captured within the discretearea. In some embodiments, the size and shape of the discrete area isadjustable according to the size, shape, or other properties of thebiological component. In some embodiments, an algorithm is used todetermine the shape and size of the discrete area. In some embodiments,the algorithm is a supervised, a self-supervised, or an unsupervisedlearning algorithm. The spatial energy modulating element may beconfigured to selectively direct the energy by, for example, inhibitingor preventing energy from being directed to one or more portions otherthan the one or more targeted portions of the fluidic device. In someembodiments, the spatial energy modulating element may comprise aphysical mask. In some cases, the spatial energy modulating element maycomprise a virtual mask. In some cases, the spatial energy modulatingelement may be a spatial light modulator (SLM). In some embodiments, theSLM is a digital micromirror device (DMD). In some embodiments, the SLMis a laser beam steered using a galvanometer. In some embodiments, theSLM is liquid-crystal based.

FIG. 24A shows a microscope equipped with a DMD and integrated UVillumination LED, according to some embodiments. FIG. 24B shows the DMDsetup projecting a virtual mask image onto the channel filled withpolymer precursor to generate a polymer matrix, according to someembodiments. FIG. 24C shows various virtual mask images that wereprojected using the DMD and the corresponding hydrogel structuresgenerated inside the fluidic device.

In certain cases, the spatial energy modulating element may beconfigured to control one or more electrodes that can selectivelyprovide energy to the one or more targeted portions of the fluidicdevice. The electrode concept may also be used to provide spatiallymodulated energy to form the hydrogel structure. In someimplementations, one or more electrodes can be arranged atpre-determined locations in the fluidic channel, thus allowing formationof the hydrogel in those locations. In alternative implementations, theelectrodes can be in the form of an array. The elements of the array canbe turned on or off on demand to create the desired spatial pattern ofenergy to form the desired shape of the hydrogels. For example, one ormore electrodes (e.g., an array of electrodes) may be disposed withinone or more portions of the fluidic device. For another example, one ormore electrodes (e.g., an array of electrodes) may be in communication(e.g., electrical communication) with one or more portions of thefluidic device.

In some embodiments, the energy source is a light generating device. Insome embodiments, the light generating device generates light at about350 nanometers to about 800 nanometers. In some embodiments, the lightgenerating device generates light at about 350 nanometers to about 400nanometers, about 350 nanometers to about 450 nanometers, about 350nanometers to about 600 nanometers, about 350 nanometers to about 800nanometers, about 400 nanometers to about 450 nanometers, about 400nanometers to about 600 nanometers, about 400 nanometers to about 800nanometers, about 450 nanometers to about 600 nanometers, about 450nanometers to about 800 nanometers, or about 600 nanometers to about 800nanometers. In some embodiments, the light generating device generateslight at about 350 nanometers, about 400 nanometers, about 450nanometers, about 600 nanometers, or about 800 nanometers. In someembodiments, the light generating device generates light at at leastabout 350 nanometers, about 400 nanometers, about 450 nanometers, orabout 600 nanometers. In some embodiments, the light generating devicegenerates light at at most about 400 nanometers, about 450 nanometers,about 600 nanometers, or about 800 nanometers. In some embodiments, thelight generating device generates UV light.

In some embodiments, a mask may prevent, or be configured to prevent,one or more portions of the energy emitting surface 210 of the energysource 203 from emitting energy (e.g., non-emitting portions 204). Insome embodiments, the mask may be a virtual mask (e.g., a computer codeor a digital system). In certain embodiments, the mask can prevent theenergy from being emitted to a location where a biological component ispresent. This may allow or permit forming a polymer matrix adjacent to,on, or encapsulating the biological component (e.g., to retain a cell,proteins, DNA molecules, RNA molecules, or other target molecules at alocation on the fluidic channel). In other embodiments, the mask mayfacilitate the polymerization such that the polymer matrix is on thebiological component. In various embodiments, the mask may be a physicalmask (e.g., an opaque material, a thermal shield, or an electromagneticshield). In some embodiments, the mask (e.g., a virtual mask or aphysical mask) can be generated using, or in combination with, adetector that detects or identifies a location of a biologicalcomponent. In some embodiments, the detector comprises a camera. In someembodiments, the detector comprises a light detector, conductivitydetector, an ultrasound detector, an ultrasonic sensor, a piezoelectricsensor, a combination thereof, or another suitable detecting device.

In some embodiments, the first surface 201 or the second surface 202 maycomprise a detector that detects, or is configured to detect, one ormore locations of one or more biological components in the fluidicdevice (e.g., in the channel 200). In certain embodiments, the energysource 203 can comprise, be coupled to, or be in communication with adetector that detects, or is configured to detect, a location of abiological component in the fluidic device. In some embodiments, thedetector may be a microscope objective for imaging the fluidic device.In various embodiments, a mask may be generated using an image obtainedfrom at least a portion of the fluidic device. The mask may allow orpermit the energy source 203 to emitting energy in or toward one or morelocations or positions where one or more biological components arepresent on or adjacent the first surface 201. The mask may inhibit orprevent the energy source 203 from emitting energy in or toward one ormore locations or positions where one or more biological components arepresent on or adjacent the first surface 201. In some embodiments, theimage may be obtained from a camera (e.g., a digital camera, fluorescentimaging camera, etc.). In some embodiments, the imaging is bright-fieldimaging, phase-contrast imaging, or fluorescence imaging, or anycombination thereof. In some embodiments, the camera may be coupled to,connected to, or in communication with the energy source 203. Forexample, the camera (not shown) may be in electrical communication withthe energy source 203. In some embodiments, the energy source 203 maycomprise the camera. In various embodiments, the energy source 203 maycomprise a microscope (e.g., a fluorescence microscope, a confocalmicroscope, lens-free imaging system, a transmission electron microscopy(TEM), a scanning electron microscope (SEM), etc.). The microscope maybe used to detect one or more positions of one or more biologicalcomponents (e.g., in combination with the detector).

In some embodiments, an algorithm is used to determine where abiological component or analyte is located based on the imaging. In someembodiments, the algorithm is a supervised, a self-supervised, or anunsupervised learning algorithm. In some embodiments, the objective iscoupled to an energy source to emit energy to the predetermined portionin the fluidic channel.

FIG. 9A shows an example of a mask comprising an energy masking region910 and an energy transparent region 915. Energy from an energy sourcemay be blocked by the energy masking region 910 to prevent the energy toform any polymer matrix in a portion of the fluidic device (e.g.,portion 920). Energy transparent region 915 may allow the energy tocommunicate with the fluidic device to form a polymer matrix 925. FIG.9B shows another example of a mask, where the energy transparent region935 is in shape of a hollow cylinder (e.g., donut). Energy being maskedby a masking region 930 may prevent energy communication with a portionof the fluidic device (e.g., a portion 940). The energy transparentregion 935 may deliver energy to the fluidic device to form a polymermatrix 945. The polymer matrix 945 may be in shape of a hollow cylinder.

FIG. 10 shows an example of biological components (i.e., indicated aswhite spots) encapsulated and/or localized using polymer matrices. Insome cases, a biological component 1001 may be localized within a hollowregion of a polymer matrix compartment 1002. In some other cases, apolymer matrix 1003 may be formed on a biological component 1004. Insome alternative cases, a polymer matrix 1005 may localize more than onebiological component. A biological compartment polymer matrix 1006 mayencapsulate one or more biological components.

In some embodiments, the fluidic device contains one or more discretelocations wherein the one or more discrete locations are not in fluidiccommunication with another discrete location. In some embodiments, theone or more discrete locations comprise the analyte. In someembodiments, the one or more discrete locations are one or more wellplates. FIG. 20 shows an example of biological components 2002encapsulated and/or localized on a well plate using polymer matrices2004. In some embodiments, the polymer matrices 2004 and biologicalcomponents 2002 are seeded on a well plate 2006. In some embodiments,the biological components are introduced to the fluidic device alongwith polymer precursors. The polymer matrices may be formed by UVphotopatterning in absence of a physical photomask. Walls 2008 mayseparate individual wells. The well plate could have any number ofwells. For example, the well plate could have 6, 12, 24, 48, or 96wells. In some embodiments, each well has a diameter of about 1millimeter to about 100 millimeters. In some embodiments, each well hasa diameter of about 1 millimeter to about 2 millimeters, about 1millimeter to about 5 millimeters, about 1 millimeter to about 10millimeters, about 1 millimeter to about 20 millimeters, about 1millimeter to about 50 millimeters, about 1 millimeter to about 100millimeters, about 2 millimeters to about 5 millimeters, about 2millimeters to about 10 millimeters, about 2 millimeters to about 20millimeters, about 2 millimeters to about 50 millimeters, about 2millimeters to about 100 millimeters, about 5 millimeters to about 10millimeters, about 5 millimeters to about 20 millimeters, about 5millimeters to about 50 millimeters, about 5 millimeters to about 100millimeters, about 10 millimeters to about 20 millimeters, about 10millimeters to about 50 millimeters, about 10 millimeters to about 100millimeters, about 20 millimeters to about 50 millimeters, about 20millimeters to about 100 millimeters, or about 50 millimeters to about100 millimeters. In some embodiments, each well has a diameter of about1 millimeter, about 2 millimeters, about 5 millimeters, about 10millimeters, about 20 millimeters, about 50 millimeters, or about 100millimeters. In some embodiments, each well has a diameter of at leastabout 1 millimeter, about 2 millimeters, about 5 millimeters, about 10millimeters, about 20 millimeters, or about 50 millimeters. In someembodiments, each well has a diameter of at most about 2 millimeters,about 5 millimeters, about 10 millimeters, about 20 millimeters, about50 millimeters, or about 100 millimeters.

One well may not be in fluidic communication with another well. Thewalls of each well 2008 may prevent fluid from traveling between thewells. In some cases, one or more assays may be conducted or performedon the biological component in a well. Different assays may be conductedor performed in different wells. For example, 6 different assays may beperformed on biological components seeded on a 6-well plate.

In some embodiments, the one or more discrete locations are open at thetop. FIG. 21 shows gel microwell structures inside 10-millimeter sizewells. The structures are open at the top, according to someembodiments. Fluorescent beads that are loaded into the fluidic devicemay also enter the wells, confirming that the wells are open at the top.

FIG. 3 is a flow chart of forming a polymer matrix on or adjacent to oneor more biological components, according to some embodiments of thepresent disclosure. The process 300 may be performed manually orautomatically (e.g., by an appropriately programmed computer system). Instep 310, a biological sample may be deposited, introduced, or providedinto at least a portion of the fluidic device. In some embodiments, amask may then be formed or generated to render one or more portions ofthe energy source directed towards a biological component non-emitting(step 320). In step 330, the energy source may apply or provide energyto at least a portion of the fluidic device. In some embodiments, theenergy source can activate or initiate polymer precursors such that thepolymer precursors form a polymer matrix (e.g., via energy provided bythe energy source). In some embodiments, an imaging of the fluidicdevice can be performed subsequent to step 310 and prior to step 320 todetermine or identify a location of the biological components togenerate a mask. In some embodiments, the mask is a virtual mask. Insome embodiments, the polymer matrix may form a compartment thatpartially or completely surrounds a biological component.

In certain cases, the energy source may be manipulated such that thepolymer matrix is formed in different steps. For example, the energysource may initiate a plurality of polymer precursors such that thepolymer precursors form an open compartment (e.g., a crescent shape orhalf-cylinder polymer matrix). The open compartment may operate tocapture and/or contain a biological component (e.g., a cell), or aportion of a sample, to a portion of the fluidic device. The orientationof the energy source or the fluidic device may be adjusted, and anadditional portion of polymer matrix may be formed. This additionalportion may be used to form one or more compartments in conjunction withthe pre-formed half-cylinder polymer matrix. In other embodimentspolymer matrix compartments can be formed in at least 2, 3, 4, 5, ormore matrix-forming steps.

FIG. 11A and FIG. 11B show an example of multi-step polymer matrixcompartment generation. FIG. 11A shows a first step of the multi-stepgeneration, where open compartments (e.g., an open compartment 1101 madefrom a polymer matrix) may be generated to capture and/or contain abiological component (e.g., a biological component 1102). A samplecomprising the biological component 1102 may have a flow direction 1103within the fluidic device (e.g., a portion of a fluidic device 1100).The open compartment 1101 may be formed by generating a polymer matrixusing an energy source and an energy modulation unit as describedherein. The open compartment may intersect a portion of the direction ofthe flow 1103 of the sample in the fluidic device. The polymer matrixopen compartment 1101 may be oblique or perpendicular to the directionof the flow 1103 of the sample in the fluidic device. FIG. 11B shows asecond step of the multi-step generation, where the open compartments(e.g., open compartment 1101) are sealed off or closed by formingpolymer matrix adjacent, around, or on the biological component (e.g.,biological component 1112). In some cases, in the second step abiological component may be completely or substantially completelyencapsulated by the polymer matrix (e.g., to form a closed compartment1111). In some cases, the polymer matrix that may form adjacent, around,or on the biological component localizes the biological component to alocation on the fluidic device 1100. Genomic and/or proteomic materialmay be extracted from the localized biological component. The polymermatrix may further localize the extracted materials. The fluidic devicemay then provide a surface where the extracted material can besequenced. In some embodiments, the extracted materials may be elutedand transferred to another device or surface for sequencing. In otherembodiments, the sequencing may be performed through short-readsequencing, nanopore sequencing, sequencing by synthesis, sequencing byin-situ hybridization, any optical readout using a microscope, or anyother suitable method of sequencing.

One or more surfaces of the fluidic device may comprise an optical(e.g., fluorescence), mechanical, electrical, or biochemical sensingelement or sensor. The sensing element may comprise a fluorescent tag,an enzyme, a primer, an oligonucleotide, or a sensor molecule (e.g., abiochemical sensor molecule). The sensing element may be used to detectand/or measure a pH, an oxygen concentration, a CO₂ concentration, orany other suitable variable. The sensing element may detect and/ormeasure a parameter locally. For example, the sensing element may detectand/or measure a pH, an oxygen concentration, or a CO₂ concentrationwithin a compartment (e.g., a polymer matrix shell cylinder) surroundingthe biological component.

Systems with Capture Elements

The present disclosure also provides systems including one or morecapture elements for immobilizing and/or compartmentalizing one or morebiological components. The system can include a fluidic device. Thefluidic device can include or contain one or more biological components.Further, the fluidic device can include or contain one or more polymerprecursors. In some embodiments, the fluidic device can include a firstsurface (e.g., in a channel and/or chamber of the fluidic device). Thefluidic device can include one or more capture elements. The captureelements can immobilize, or be configured to immobilize, at least one ofthe one or more biological components at a location on or adjacent tothe first surface (or any suitable surface). Immobilization or couplingof a biological component to a capture element can form an immobilizedbiological component. The system may further include at least one energysource in communication with the fluidic device. In certain embodiments,the at least one energy source can provide or supply energy, or beconfigured to provide or supply energy, to at least a portion of thefluidic device. Accordingly, the energy source can activate or cause theone or more polymer precursors (e.g., disposed in the fluidic device) toform at least one polymer matrix on or adjacent to an immobilizedbiological component. In various embodiments, the fluidic device mayfurther include a platform or a stage to hold the fluidic device. Insome embodiments, the system may also include a sequencing device (e.g.,a next-generation sequencing device) to obtain sequencing data. Thepolymer matrix formed in the fluidic device may be used to capture andlocalize a biological component. Genomic and/or proteomic material maybe extracted using the fluidic device. The fluidic device may thenprovide a surface where the extracted material can be sequenced. In someembodiments, the extracted materials may be eluted and transferred toanother device or surface for sequencing. In other embodiments, thesequencing may be performed through short-read sequencing, nanoporesequencing, sequencing by synthesis, sequencing by in-situhybridization, or any optical readout using a microscope.

In order to immobilize a biological component, a fluidic device maycomprise one or more capture sites. A capture site may include a captureelement. In some embodiments, the one or more capture elements or sitesmay comprise or be disposed in a pattern. A fluidic device may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400,500, 600, 700, 1,000, 10,000, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10²⁰capture elements, or any number of capture elements between any of thetwo numbers mentioned herein. In some embodiments, the fluidic devicemay comprise more than 10²⁰ capture elements.

The fluidic device may comprise a channel. The fluidic device maycomprise a chamber. FIG. 4A shows an example of at least a portion of achannel 400 in a fluid device. One or more capture elements 411 may bedisposed or positioned on a first surface 401 of the fluidic device. Insome cases, a second surface 402 may comprise one or more captureelements. The capture elements may be disposed on both surfaces or anyother suitable surface. A capture element may comprise or be at leastpartially formed by a functional group. Some non-limiting examples offunctional groups include a capture reagent (e.g.,pyridinecarboxaldehyde (PCA)), a biotin, a streptavidin, a strep-tag II,a linker, or a functional group that can react with a molecule (e.g., analdehyde, a phosphate, a silicate, an ester, an acid, an amide, analkyne, an azide, or an aldehyde dithiolane). The functional group maycouple specifically to an N-terminus or a C-terminus of a peptide. Thefunctional group may couple specifically to an amino acid side chain.The functional group may couple to a side chain of an amino acid (e.g.,the acid of a glutamate or aspartate, the thiol of a cysteine, the amineof a lysine, or the amide of a glutamine or asparagine). The functionalgroup may couple specifically to a reactive group on a particularspecies, such as a membrane-bound molecule on a cell (e.g., aglycoprotein of a eukaryotic cell or a pilus on a plasma of aprokaryote). In some examples, the capture elements can comprisefibronectin. In another example, the capture elements can comprise RGDpeptides. In some cases, capture elements may comprise antibodies. Insome examples, the functional motif can be reversibly coupled andcleaved (e.g., by using an enzyme). FIG. 4B illustrates an example of abiological component 51 in contact with or coupled to a capture element411. In some cases, a repelling surface coating (e.g., PEG) may be usedto prevent the polymer matrix form covering or trapping a biologicalcomponent.

In various instances, the capture element may comprise a physical trap,a hydrodynamic trap, a geometric trap, a well, an electrochemical trap(e.g., trapping charge molecules), streptavidin, an antibody, anaptamer, affinity binding (e.g., a peptide that may bind to a surfaceprotein of a cell), one or more magnetic material (e.g., magnetic disk,magnetic array, or magnetic particles), a dielectrophoretic trap (e.g.,electrode array), or a combination thereof. The trap may comprise apolymer matrix or hydrogel. The polymer matrix or hydrogel trap may beconstructed or deconstructed on demand using an energy source and/ordegradation similar to the polymer matrix compartments mentioned herein.For example, a capture element may comprise a well. The well may be from1 μm to 50 μm in diameter. In some embodiments, the well may be from 1μm to 20 μm, 20 μm to 30 μm, 30 μm to 40 μm, or 40 μm to 50 μm indiameter. The well may be more than 50 μm in diameter. The well may beless than 1 μm in diameter. In some embodiments, the well may be from0.1 μm to 100 μm in depth. In certain embodiments, the well may be morethan 100 μm in depth. The well may be less than 0.1 μm in depth. Thedepth of the well may be from 0.1 μm to 0.5 μm, 0.1 μm to 1 μm, 0.1 μmto 5 μm, 0.1 μm to 10 μm, 0.1 μm to 20 μm, 0.1 μm to 30 μm, 0.1 μm to 50μm, 0.1 μm to 100 μm, 0.5 μm to 1 μm, 0.5 μm to 5 μm, 0.5 μm to 10 μm,0.5 μm to 20 μm, 0.5 μm to 30 μm, 0.5 μm to 50 μm, 0.5 μm to 100 μm, 1μm to 5 μm, 1 μm to 10 μm, 1 μm to 20 μm, 1 μm to 30 μm, 1 μm to 50 μm,1 μm to 100 μm, 5 μm to 10 μm, 5 μm to 20 μm, 5 μm to 30 μm, 5 μm to 50μm, 5 μm to 100 μm, 10 μm to 20 μm, 10 μm to 30 μm, 10 μm to 50 μm, 10μm to 100 μm, 20 μm to 30 μm, 20 μm to 50 μm, 20 μm to 100 μm, 30 μm to50 μm, 30 μm to 100 μm, or 50 μm to 100 μm. The depth of the well may beabout 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about20 μm, about 30 μm, about 50 μm, or about 100 μm. The depth of the wellmay be at least 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, or 50μm. The depth of the well may be at most 0.5 μm, 1 μm, 5 μm, 10 μm, 20μm, 30 μm, 50 μm, or 100 μm.

In some embodiments, the fluidic device may comprise a repelling surfacecoating that may be used to prevent capturing of a biological componentat predefined locations. FIG. 12A illustrates a portion of a surface1201 of a fluidic device, where the surface 1201 may comprise acapturing site 1202 and a repelling site 1203. The surface 1201 may befunctionalized using a surface coating (e.g., PEG) to generate therepelling site 1203. The repelling site 1203 may prevent biologicalcomponents from binding to the surface 1201 at the location of therepelling site 1203 and drive the biological component to the capturesite 1202. In some cases, the surface 1201 may only comprise therepelling sites without a capturing site. The repelling site mayinitially localize the biological components. A polymer matrix may beformed by directing an energy source to the repelling sites to formcompartments adjacent to the biological components that may be locatedin between the repelling sites. FIG. 12B shows an example of biologicalcomponents contained in predefined locations in the fluidic device. FIG.12C shows a higher magnification example of biological componentscontained in predefined locations in the fluidic device.

An energy source may be used to form a polymer matrix on, around, oradjacent to at least a portion of a captured biological component. Insome embodiments, a mask may be used to allow or permit the energysource to direct energy toward a location or position of a capturedbiological component. In certain embodiments, a mask may be used toinhibit or prevent the energy source from directing energy toward alocation or position of a captured biological component. The mask may beconfigured to direct the energy to predetermined or selected locationsto form a polymer matrix surrounding or at least partially surroundingthe one or more biological components. The mask may be generated basedat least in part on a pattern of the capture sites (e.g., the pattern ofthe capture sites/elements on a surface of a fluidic device). In someembodiments, the mask may be configured to prevent energy from beingdirected to a location surrounding a capture site or element which hasnot captured or coupled a biological component. In certain cases, toanalyze a single cell, the mask may be configured to prevent the energyfrom being emitted adjacent to a location of a capture element which hascaptured or coupled two or more biological components. In someembodiments, the mask may be configured to allow or permit energy to beemitted adjacent to a location of a capture element which has capturedtwo or more biological components, for example, to allow analysis ofcell-cell interactions. In certain embodiments, the mask may be aphotolithographic mask or another suitable mask, as described herein. Insome embodiments, the system may further comprise a detector, forexample, to detect a location of a biological component, as describedherein. The mask may be generated based at least in part on the detectedlocation of a biological component. Additionally, the mask mayselectively direct or supply energy from the energy source to thefluidic device, as described herein.

FIG. 4C illustrates an example of a method of forming polymer matricesadjacent to (e.g., surrounding) biological components. A polymer matrix408 may be formed adjacent to a capture element 411. The polymer matrix408 may be configured to hold a biological component 51 in place orwithin an analysis chamber or compartment 420. The compartment 420 maybe formed, at least in part, by the polymer matrix 408, the firstsurface 401, and the second surface 402 forming a chamber or at leastpartially sealed-off space within the fluidic device (e.g., around thebiological component 51). In some embodiments, the polymer matrix 408may form the compartment 420 surrounding the biological component 51.The compartment 420 may hold the biological component 51 in place. Thepolymer matrix 408 and/or the compartment 420 may inhibit or prevent acompound associated with the biological component 51 from leaving thecompartment. In some embodiments, the compound associated with thebiological component may comprise a nucleic acid (e.g., DNA or RNA), aprotein, a metabolite, an enzyme, an antibody, combinations thereof, orany other suitable compound or material. In some embodiments, thesurface of the polymer matrix or hydrogel may be functionalized bycoupling a functional group to the polymer matrix or hydrogel. Thefunctionalized surface of the polymer matrix inside the compartment maybe coupled to a capturing element (e.g., an antibody) to capture amolecule secreted by the biological component (e.g., secreted protein).The capturing element or the captured molecule may then be read out by asensing molecule or by a labeling method, for example, by fluorescentlabeling. In some embodiments, a polymer matrix may be configured toallow passage of one or more compounds associated with a biologicalcomponent. In some embodiments, a polymer matrix may be configured toallow passage of a reagent. The reagent may comprise, for example, oneor more enzymes, chemicals, oligonucleotides (e.g., one or more primershaving a size of less than 50 base pairs), lysozymes, proteinase K,random hexamers, polymerases, transposases, ligases, catalyzing enzymes,deoxynucleotide triphosphates, buffers, cell culture media, divalentcations, combinations thereof, or any other suitable reagent.

The pore size in the polymer matrix may be modulated using a chemicalreagent, or by applying heat, electricity, light, or another suitablestimulus. In other words, the polymer matrix may comprise tunableproperties (e.g., the pore size) In some cases, the polymer matrix maycomprise a thermoresponsive or temperature-responsive polymer. Athermoresponsive polymer (e.g., poly(N-isopropylacrylamide) (NIPAAM))may phase separate from a solution upon heating or upon cooling (e.g.,polymer showing lower critical solution temperature (LCST) or uppercritical solution temperature (UCST). The polymer matrix may comprisepolymer which may collapse at high temperature in order to, for example,control the pore size of the hydrogel or polymer matrix. Non-limitingexamples of thermoresponsive polymers that may be used to formhydrogel/polymer matrix with tunable properties may include Poly(N-vinylcaprolactam), Poly(N-ethyl oxazoline), Poly(methyl vinyl ether),Poly(acrylic acid-co-acrylamide), or a combination thereof. A change intemperature may close or open a pore in the polymer matrix to allow areagent, a nucleic acid molecule, a protein, or any biomolecule ormolecule smaller than the pore size to be released from the polymermatrix compartment. In some cases, the released molecule may be amolecule of interest. The released molecule may be collected foranalysis. The pore size may be decreased after releasing the molecule(s)to localize the biological component and other molecules within thepolymer matrix. In some cases, the remaining localized molecules may bea molecule(s) of interest. For example, the pore size may be adjusted toallow a protein tag (e.g., shorter DNA oligomers) to be released fromthe polymer matrix compartment and the fluidic chambers. The protein tagmay then be collected while the mRNA remain localized within the polymermatrix compartment for further analysis.

The polymer matrix may have a pore size of about 5 nanometers (nm) toabout 100 nm. The polymer matrix may have a pore size of about 5 nm toabout 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 nm toabout 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm,about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm toabout 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm,about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm toabout 90 nm, about 10 nm to about 100 nm, about 10 nm to about 110 nm,about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm toabout 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm,about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm toabout 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm,about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm toabout 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm,about 30 nm to about 100 nm, about 30 nm to about 110 nm, about 40 nm toabout 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm,about 40 nm to about 80 nm, about 40 nm to about 90 nm, about 40 nm toabout 100 nm, about 40 nm to about 110 nm, about 50 nm to about 60 nm,about 50 nm to about 70 nm, about 50 nm to about 80 nm, about 50 nm toabout 90 nm, about 50 nm to about 100 nm, about 50 nm to about 110 nm,about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm toabout 90 nm, about 60 nm to about 100 nm, about 60 nm to about 110 nm,about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm toabout 100 nm, about 70 nm to about 110 nm, about 80 nm to about 90 nm,about 80 nm to about 100 nm, about 80 nm to about 110 nm, about 90 nm toabout 100 nm, about 90 nm to about 110 nm, or about 100 nm to about 110nm. The polymer matrix may have a pore size of about 5 nm, about 10 nm,about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about70 nm, about 80 nm, about 90 nm, about 100 nm, or about 110 nm. Thepolymer matrix may have a pore size of at least about 5 nm, about 10 nm,about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about70 nm, about 80 nm, about 90 nm, about 100 nm, or less. The polymermatrix may have a pore size of at most about 10 nm, about 20 nm, about30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm,about 90 nm, about 100 nm, about 110 nm, or more.

A polymer matrix may be degradable. In some embodiments, the degradablepolymer matrix may be depolymerized (e.g., into polymer precursors). Atleast a portion of the polymer precursors from a degraded polymer matrixmay be used again to form another polymer matrix. An energy source asprovided herein may be used to depolymerize a polymer matrix. In someembodiments, by depolymerizing a polymer matrix, a compartmentsurrounding a biological component may be deconstructed (e.g., torelease the biological component). In some embodiments, releasing thebiological component may also include releasing a biological componentfrom a captured element. That is, the biological component may bedetached or uncoupled from the capture element. In some embodiments, acapture element, or a portion of a capture element, may be cleaved usinga chemical compound (e.g., an agarase, a dextranase, ametalloproteinase, or other enzyme) or by providing energy to thecapture site or capture element using the energy source (e.g., lightmediated degradation). In some cases, the capture element may be cleavedusing hydrolysis, ester hydrolysis, enzymatic hydrolysis, reversibleclick reactions, or photolytic cleaving. In some cases, the captureelement may be uncoupled from a surface or a biological component byapplying physical force (e.g., sonication, agitating the fluidic device,etc.). In some cases, a capture element may comprise agarose which maybe degraded or cleaved using an agarase. In some cases, a captureelement may comprise dextran which may be degraded or cleaved usingdextranase. In some cases, a capture element may comprise ametalloproteinase (MMP) degradable peptide. In certain cases, thebiological component may be decoupled from a capture element using achemical method (e.g., using a digesting enzyme to cleave a bond) or aphysical method (e.g., using an energy source to provide heat,microwave, electromagnetic waves, electromagnetic field, sonic waves,etc.).

In some embodiments, a polymer matrix may be contacted with a cleavagemix to degrade or depolymerize the polymer matrix. In some embodiments,the cleavage mix may comprise dithiothreitol (DTT), β-mercaptoethanol,glutathione, tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments,heat may be directed to a polymer matrix to degrade or depolymerize thepolymer matrix. In some embodiments, the polymer matrix may be heated toat least 90° C. In some embodiments, the polymer matrix may be heated tofrom 80° C. to 100° C., 90° C. to 110° C., 110° C. to 120° C., 120° C.to 180° C., or 180° C. to 250° C. A wavelength of light appropriate tocleave a photocleavable crosslinker that crosslinks a polymer matrix maybe directed to a polymer matrix to degrade or depolymerize the polymermatrix. In some embodiments, the fluidic device may comprise aphotoactive compound (e.g., a photoacid generator or a photobasegenerator) that can degrade or depolymerize the polymer matrix uponexposure to light energy (e.g., a wavelength of light).

The polymer matrix may comprise a hydrogel. In some embodiments, thepolymer matrix may form a wall, where the polymer matrix wall may becoupled to a first surface and/or the second surface. In someembodiments, the wall may be a hydrogel wall. The polymer matrix wall orthe hydrogel wall may have a thickness from 1 μm to 250 μm. The polymermatrix wall or hydrogel wall may have a thickness from 1 μm to 5 μm, 1μm to 10 μm, 1 μm to 20 μm, 1 μm to 30 μm, 1 μm to 40 μm, 1 μm to 50 μm,1 μm to 100 μm, 1 μm to 150 μm, 1 μm to 250 μm, 5 μm to 10 μm, 5 μm to20 μm, 5 μm to 30 μm, 5 μm to 40 μm, 5 μm to 50 μm, 5 μm to 100 μm, 5 μmto 150 μm, 5 μm to 250 μm, 10 μm to 20 μm, 10 μm to 30 μm, 10 μm to 40μm, 10 μm to 50 μm, 10 μm to 100 μm, 10 μm to 150 μm, 10 μm to 250 μm,20 μm to 30 μm, 20 μm to 40 μm, 20 μm to 50 μm, 20 μm to 100 μm, 20 μmto 150 μm, 20 μm to 250 μm, 30 μm to 40 μm, 30 μm to 50 μm, 30 μm to 100μm, 30 μm to 150 μm, 30 μm to 250 μm, 40 μm to 50 μm, 40 μm to 100 μm,40 μm to 150 μm, 40 μm to 250 μm, 50 μm to 100 μm, 50 μm to 150 μm, 50μm to 250 μm, 100 μm to 150 μm, 100 μm to 250 μm, or 150 μm to 250 μm.The polymer matrix wall or hydrogel wall may have a thickness of about 1μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm,about 50 μm, about 100 μm, about 150 μm, or about 250 μm. The polymermatrix wall or hydrogel wall may have a thickness of at least 1 μm, 5μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 150 μm, 250 μm, or more.The polymer matrix wall or hydrogel wall may have a thickness of at most5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 150 μm, or 250 μm. Thepolymer matrix wall or hydrogel wall may have a thickness of less than 1μm.

In some embodiments, a hydrogel may comprise polyethylene glycol(PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine, PEG,polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, bisacrylamide, diacrylate, diallylamine,triallylamine, divinyl sulfone, diethyleneglycol diallyl ether,ethyleneglycol diacrylate, polymethyleneglycol diacrylate,polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate,ethoxylated trimethylol triacrylate, ethoxylated pentaerythritoltetraacrylate, or combinations or mixtures thereof. In some embodiments,the hydrogel may comprise PEG-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/PPO. For example,a PEG molecule may comprise a multi-arm PEG derivative with thiol groupsat each terminal of the arms connected to one pentaerythritol core. Areactive free thiol, SH, sulfhydryl, or mercapto group may selectivelyreact with the microfluidic surface (e.g., a maleimide or a transitionmetal surface) including gold, silver, etc. PEG-SH can be air oxidizedto form S—S disulfide (disulfide) bonds, which can be reversed withreducing agents to form reversible PEGylation or PEG hydrogel.

As discussed, the polymer matrix (e.g., hydrogel) may be porous enoughto allow passage of a reagent (e.g., an enzyme, a reagent, a smallmolecule, an antibody), while preventing passage of the capturedbiological component (e.g., DNA, RNA, a protein, cells, etc.) or acompound associated with the biological component (e.g., DNA, RNA, anantibody, a secreted compound from a cell, etc.). In some embodiments, areagent may comprise an enzyme, or a primer having a size of less than50 base pairs (bp). A primer may have a size from 5 pb to 50 bp. In someembodiments, a primer may have a size from 5 pb to 10 bp, 10 bp to 20bp, 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In certainembodiments, a primer may have a size of more than 50 bp. In variousembodiments, a primer may have a size of less than 5 bp.

In certain embodiments, a first surface, a second surface, or bothsurfaces of a channel in the fluidic device may be functionalized, asdescribed herein. A surface (e.g., a first surface, a second surface, athird surface, etc.) of the fluidic device may comprise a compoundconfigured to bind to a biological component (e.g., a capturedbiological component). In some embodiments, a surface (e.g., the firstsurface, the second surface, the third surface, etc.) of the fluidicdevice may comprise one or more barcodes. One or more surfaces maycomprise oligos to from DNA clusters for sequencing. In some cases, oneor more surfaces may comprise one or more nanopore readers for directDNA and/or RNA readout. One or more surfaces may comprise nanowells tocapture single RNA molecules and/or single DNA molecules or to contain aDNA/RNA library. In some alternative cases, one or more surfaces maycomprise patterned hydrophobic/hydrophilic features for selectivedeposition of DNA nanoballs. Nanoballs may be generated bycircularization and amplification of DNA libraries from DNA/RNAmolecules.

One or more surfaces of the fluidic device may comprise an optical(e.g., fluorescence), mechanical, electrical, or biochemical sensingelement or sensor. The sensing element may comprise a fluorescent tag,an enzyme, a primer, an oligonucleotide, or a sensor molecule (e.g., abiochemical sensor molecule). The sensing element may be used to detectand/or measure a pH, an oxygen concentration, a CO₂ concentration, orany other suitable variable. The sensing element may detect and/ormeasure a parameter locally. For example, the sensing element may detectand/or measure a pH, an oxygen concentration, or a CO₂ concentrationwithin a compartment (e.g., a polymer matrix shell cylinder) surroundingor encapsulating the biological component.

Multi-Tiered Systems

Also provided herein are systems for analyzing a biological componentcomprising at least a flow channel (e.g., a first or upper layer) and ananalysis channel (e.g., a second or lower layer). The system maycomprise a fluidic device including a flow channel, an analysis channel,and a layer or wall disposed between the flow channel and the analysischannel. The system may include at least one energy source incommunication with the fluidic device, as described herein. The analysischannel may be disposed adjacent to the flow channel, where at least oneflow inhibition element may be disposed within the flow channel toinhibit or stop flow of the biological component in the flow channel.The layer disposed between the flow channel and the analysis channel maycomprise at least one sealable aperture disposed at or adjacent to theat least one flow inhibition element. One or more biological componentsmay be stopped or trapped adjacent to the sealable aperture. The atleast one sealable aperture may be configured to allow passage of theone or more biological components. For example, the sealable aperturemay be configured to allow passage of the one or more biologicalcomponents from the flow channel to the analysis channel. The at leastone energy source may be in communication with the analysis channel.Furthermore, the at least one energy source may form, or be configuredto form, a polymer matrix within the analysis channel.

As described herein, in some embodiments, the fluidic device maycomprise a microfluidic device or a nanofluidic device. In certainembodiments, the fluidic device may be used for nucleic acid sequencing.In some cases, the fluidic device may comprise a nucleic acid sequencingflow cell. In other cases, sequencing may comprise short-readsequencing, nanopore sequencing, sequencing by synthesis, sequencing byin-situ hybridization, sequencing through collection of any opticalreadouts, or any other suitable method of sequencing. As describedherein, the biological component may comprise a cell, a cell lysate, anucleic acid, a microbiome, a protein, a mixture of cells, aspatially-linked biological component, a metabolite, a combinationthereof, or any other suitable biological component. In some cases, themixture of cells may comprise two or more different cell types. Forexample, the mixture of cells may comprise a first cell type and asecond cell type. In some cases, the mixture of cells may comprise 2, 3,4, 5, 6, 7, 8, 9, 10, or more cell types. A cell may be a mammalian cell(e.g., a human cell), a fungal cell, a bacterial cell, a tumor spheroid,a combination thereof, or any other suitable cell. In some cases, thebiological component may comprise a tumor spheroid or a spatially-linkedbiological component (or sample).

In some cases, the nucleic acid may comprise at least 100 bases or basepairs. In certain embodiments, a nucleic acid comprises a DNA or an RNA.The DNA may be at least 100 bp long. In some embodiments, the DNA mayinclude at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp,700 bp, 800 bp, 900 bp, 1,000 bp, 10 kilo base pairs (kbp), 100 kbp, 1mega base pair (Mbp), 100 Mbp, 1 giga base pair (Gbp), 10 Gbp, 100 Gbp,or more base pairs. The biological component may comprise a DNA moleculethat comprises any number of base pairs in between the mentioned numbersherein. For example, the DNA may comprise from 50 bp to 1,000 bp, 300 bpto 10 kbp, or 1,000 bp to 10 Gbp. The RNA may be dsRNA. The dsRNA maycomprise at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp,700 bp, 800 bp, 900 bp, 1,000 bp, 10 kbp, or 100 kbp. The biologicalcomponent may comprise a dsRNA molecule that includes any number of basepairs in between the mentioned numbers herein. For example, the dsRNAmay comprise from 50 bp to 1,000 bp, 300 bp to 10 kbp, or 1,000 bp to100 kbp. The RNA may be ssRNA. The ssRNA may comprise at least 50nucleotides to 100,000 nt. The ssRNA may comprise from 50 nt to 100 nt,50 nt to 1,000 nt, 50 nt to 10,000 nt, 50 nt to 100,000 nt, 100 nt to1,000 nt, 100 nt to 10,000 nt, 100 nt to 100,000 nt, 1,000 nt to 10,000nt, 1,000 nt to 100,000 nt, or 10,000 nt to 100,000 nt. In some cases,the ssRNA may be less than 50 nucleotides long. The ssRNA may be morethan 100,000 nucleotides long.

In some embodiments, the flow channel or a portion thereof may beparallel, or substantially parallel, with the analysis channel or atleast a portion thereof. In some embodiments, the flow channel may beremovably couplable to the analysis channel. For example, a user mayremove the flow channel from the analysis channel. Accordingly, aportion of the fluidic device comprising the analysis channel may beused to conduct various analyses or experiments. With the portion of thefluidic device comprising the flow channel removed, the portion of thefluidic device comprising the analysis channel may be more accessible,e.g., to detectors, cameras, or other devices for analyzing thebiological components within the analysis channel.

In some cases, the analysis channel may include polymer matrixstructures for capturing or trapping a biological component or amolecule or compound produced by the biological component (e.g., priorto introduction of the biological components into the fluidic device).For example, a user may obtain an analysis channel that includes polymermatrix structures. That is, the user may not form the polymer matrixstructures. In various cases, the analysis channel may be configured toinclude polymer matrix structures for capturing or trapping a biologicalcomponent or a molecule or compound produced by the biologicalcomponent. For example, in such embodiments, subsequent to introductionof the biological components into the fluidic device and the analysischannel, one or more polymer matrix structures may be formed in theanalysis channel. The analysis channel may be configured for a screeningprocess, a library preparation, or another suitable process. In someembodiments, the screening process may be for drug screening, antibioticscreening, culture conditions screening, or CRISPR screening. In certaincases, a plurality of samples may be placed into a plurality ofchannels. The plurality of samples may be screened against a variety ofconditions in other signal-containing channels.

The sealable aperture may be configured to transition from a sealedstate to an open state. For example, a sealable aperture may comprise aheat sensitive polymer that can melt, for example, upon receiving heatand render the sealable aperture open. In some cases, the passage of thebiological component through the sealable aperture may be inhibited inthe sealed state. In certain cases, the passage of the biologicalcomponent through the sealable aperture may be allowed in the openstate. In some cases, the sealable aperture may be sealed with anagarose gel, a temperature-soluble polymer, an N-isopropylacrylamide(NIPAAm) polymer, a wax compound, an alginate, or any other suitablecompound or material.

FIGS. 6A and 6B show a portion of a fluidic device configured to trap abiological component 50. The fluidic device may comprise a flow channelor chamber 651, an analysis channel or chamber 652, and a layer or wall653 disposed between at least a portion of the flow channel 651 and theanalysis channel 652. The layer 653 may comprise one or more sealableapertures or openings 654. Additionally, one or more flow inhabitationelements 655 may inhibit or prevent, or be configured to inhibit orprevent, the biological component 50 from flowing along the flow channel611. A flow inhibition element 655 may be configured to stop or trap thebiological component 50 adjacent to a sealable aperture 654. Asdescribed herein, the sealable aperture 614 may be configured totransition from a sealed state (e.g., a closed state) or configurationto an open state or configuration. FIG. 6A shows an example of thesealable aperture 614 in a sealed state. FIG. 6B shows an example of thesealable aperture 654 in an open state. Upon transitioning to a sealedstate to an open state, the sealable aperture 654 may allow or permitpassage of the biological component 50 from at least a portion of theflow channel 651 to at least a portion of the analysis channel 652. Incertain instances, the analysis channel 652 may be placed, or configuredto be placed, below the flow channel 651 to allow the biologicalcomponent 50 to be transferred to the analysis channel 652 from the flowchannel 651 by a force provided (e.g., via gravity, high pressure pulseby pressurizing a flow in the flow channel, and generating negativepressure in the analysis channel). In some embodiments, the fluidicdevice may be spun or centrifuged to disposed the one or more biologicalcomponents from the flow channel to the analysis channel. Reagents canbe disposed or passed through at least a portion of the analysis channel652, for example, to conduct analyses or experiments are providedherein.

As shown in FIG. 6A, the flow inhibition element 655 may be disposedwithin at least a portion of the flow channel 651 to inhibit or preventflow of a biological component (e.g., biological component 50) in theflow channel 651. The flow inhibition element 655 may be configured tocapture or trap the biological component 50 in at least a portion of theflow channel 651. In some cases, the flow inhibition element 655 mayextend from a surface (e.g., surface 669) of the flow channel 651. Insome cases, the surface 669 may be disposed opposite of a flow channelsurface 661, which is adjacent to the layer 653.

In various cases, the analysis channel 652 may comprise a surface 659disposed opposite of the analysis channel surface 663, which is adjacentto or a surface of the layer 653. The analysis channel 652 may compriseone or more polymer matrices 656. The analysis channel 652 may compriseone or more polymer precursors. For example, one or more polymerprecursors may be disposed in at least a portion of the analysis channel652. The one or more polymer matrices 656 may be formed using an energysource which provides energy to the one or more polymer precursors inthe analysis channel 602. The energy source may be in opticalcommunication, electrochemical communication, electromagneticcommunication, thermal communication, or microwave communication withthe fluidic device or the analysis channel 652. In some cases, theenergy source may be a light generating device, a heat generatingdevice, an electrochemical generating device, an electrode, a microwavedevice, or a combination thereof. The energy source may selectivelyprovide energy to the analysis channel 652 to form polymer matrices atpredefined locations. A spatial energy modulating element may be used toselectively provide energy to the analysis channel 652.

In some cases, the spatial energy modulating element may comprise aphotolithographic mask, a DMD system, or other suitable mask. The one ormore polymer matrices 656 may be formed before the sealable aperture 654transitions to an open state (e.g., as shown in FIG. 6A). For example, apolymer matrix may be formed and aligned with the sealable aperture suchthat the biological component 650 held by the inhibition element 655 maydirected (e.g., fall by gravity or by fluid pressure) into a compartment620 when the sealable aperture 654 is rendered open. The one or morepolymer matrices 656 may be formed after the sealable aperture 654transitions to an open state (e.g., as shown in FIG. 6B). The one ormore polymer matrices 656 may form an analysis chamber or compartment620, as described herein.

FIGS. 7A and 7B show a top view of a fluidic device. A flow inhibitionchannel 675 may be configured to inhibit a biological component 20 fromflowing along a flow channel 651. Flow of a fluid (e.g., a fluidincluding the biological component) through the flow channel 651 and theflow inhibition channel 651 may cause the biological component 20 to betrapped or stopped at an opening of the flow inhibition channel 675 asdepicted in FIG. 7A. As illustrated, a dimension (e.g., a width) of theflow inhibition channel 675 may be too small or narrow to allow orpermit passage of the biological component 20 through the flowinhibition channel 675. As shown in FIG. 7B, a polymer matrix 676 may beformed on or adjacent to (e.g., surrounding) the biological component20. In some cases, the polymer matrix may surround at least a portion ofthe biological component. The fluidic device of FIGS. 7A and 7B may be asingle-layer fluidic device. That is, the polymer matrix may be formedin the flow channel 651. As illustrated, a path of the flow channel 651may be circuitous. For example, the flow channel 651 may include one ormore curves. In some embodiments, the path of the flow channel may bestraight, substantially straight, in a zig-zag pattern, or any othersuitable shape.

In certain embodiments, the fluidic device of FIGS. 7A and 7B maycomprise two or more layers. For example, the fluidic device may includea flow channel and an analysis channel (similar to the system shown inFIGS. 6A and 6B). Further, a sealable aperture may be disposed at oradjacent to a portion of a flow inhibition channel. In such embodiments,the biological component may be transferred into the analysis channel(e.g., disposed adjacent to or below the flow channel) through asealable aperture, as described herein. In some cases, the analysischannel may receive two or more biological components. For example, theanalysis channel may receive 2, 3, 4, 5, 6, 7, 8, 9, 10, or morebiological components.

FIG. 8 shows an example of a fluidic device including, or configuredfor, a plurality of reagents and/or analytes (R1, R2, R3, and R4). Thefluidic device may comprise a first flow channel 851 a to receive one ormore biological components from a first sample. The first flow channel851 a may allow or permit flow or passage of one or more biologicalcomponents from the first sample. Further, the first flow channel 851 amay allow or permit flow or passage of one or more polymer precursors.The fluidic device may comprise a second flow channel 851 b to receiveone or more biological components from a second sample. The second flowchannel 851 b may allow or permit flow or passage of one or morebiological components from the second sample. Further, the second flowchannel 851 b may allow or permit flow or passage of one or morebiological components from the second sample.

The first flow channel 851 a and/or the second flow channel 851 b maycomprise a plurality of inhibition elements (e.g., inhibition element855). A biological component (e.g., biological component 50) may betrapped or localized by the inhibition element 855. As described herein,the first flow channel 851 a and/or the second flow channel 851 b maycomprise one or more sealable apertures disposed at or adjacent to theone or more inhibition elements 855 that can be opened (e.g.,transitioned from a sealed state to an open state) to allow thebiological component to move into a first analysis channel 852 a or asecond analysis channel 852 b. The first and second flow channels 851 a,851 b may be disposed above the first and second analysis channels 852a, 852 b (e.g., in an upper layer and a lower layer similar to thefluidic device illustrated in FIGS. 6A and 6B). A polymer matrix 856 maybe formed surrounding the biological component 50. The polymer matrix856 may partially surround the biological component 50. The polymermatrix 856 may form a compartment or an analysis chamber 820 to localizethe biological component 50 within at least a portion of an analysischannel (e.g., analysis channels 851 a, 851 b).

The first analysis channel 852 a may comprise one or more reagentsand/or analytes that are different from the one or more reagents and/oranalytes in the second analysis channel 852 b. The first analysischannel 852 a may comprise one or more reagents and/or analytes that arethe same as the one or more reagents and/or analytes in the secondanalysis channel 852 b. In some cases, the fluidic device may include atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, or more flow channels Incertain cases, the fluidic device may include at least 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 25, 50, or more analysis channels. The fluidic device mayanalyze a plurality of biological components in parallel. The pluralityof biological components may be exposed to one or more differentreagents and/or analytes, as provided herein. Such a configuration(e.g., as shown in FIG. 8) may allow a plurality of biologicalcomponents in one or more samples to be analyzed under variousconditions provided by the different reagents and/or analytes. Thefluidic device, shown in FIG. 8, may be used for a screening process.The screening process may be for drug screening, antibiotic screening,culture conditions screening, or CRISPR screening. The screening processmay be performed in combinatorial manner. For example, a plurality ofsamples may be loaded in a plurality of flow channels (e.g., inparallel) which may be screened against a plurality of conditions in theplurality of analysis channels.

The first and/or the second samples may be homogenous or heterogenous.For example, the one or more biological components in the first samplemay be the same or different. The first sample may be different from thesecond sample. In some cases, a biological component may be releasedfrom a compartment or analysis chamber 820 by selectively degrading apolymer matrix, as described herein. In other words, a polymer matrixmay be degraded “on demand” (e.g., by a user or as directed by acomputer). In various embodiments, the degradation may be achievedthrough the use of localized stimuli. In certain embodiments, thedegradation may be achieved through the use of heat, light,electrochemical reactions, or some combination thereof. The releasedbiological component may be collected using an outlet channel (e.g.,outlet channel 881 a or 881 b).

As described in reference to the fluidic device of FIGS. 6A and 6B, alayer may be disposed between the flow channels 851 a, 851 b and theanalysis channels 852 a, 852 b. The analysis channel surface adjacent tothe layer (e.g., similar to surface 661 shown in FIG. 6A), the analysischannel surface opposite of the layer (e.g., similar to surface 659shown in FIG. 6A), or both may comprise one or more barcodes, asdescribed herein.

In some cases, the channels (e.g., channels 100, 200, 400) and/or theanalysis channels (e.g., analysis channels 652, 852 a, 852 b) maycomprise molecules in addition to, or instead of, the one or morebarcodes. For example, any of the surfaces of the one or more channelsand/or analysis channels may comprise an optical (e.g., fluorescence),mechanical, electrical or biochemical sensing element or sensor. Thesensing element may comprise a fluorescent tag, an enzyme, a primer, anoligonucleotide, or a sensor molecule (e.g., a biochemical sensormolecule). The sensing element may be used to detect and/or measure apH, an oxygen concentration, a CO₂ concentration, or any other suitablevariable. The sensing element may detect and/or measure a parameterlocally. For example, the sensing element may detect and/or measure apH, an oxygen concentration, or a CO₂ concentration within a compartment(e.g., a polymer matrix shell cylinder) surrounding the biologicalcomponent.

FIG. 16A shows a portion of any of a system as provided herein includingan energy source 1610 (e.g., a light source) and a spatial energymodulating element (e.g., a mask) 1630. In some embodiments, the energysource 1610 may be coupled to an objective (e.g., a microscope objectiveor lens). The energy source 1610 may emit energy as electromagneticwaves (e.g., microwaves, light, heat, etc.). In some embodiments, theemitted energy 1620 may form, or may be sufficient to form, a polymermatrix 1660 within at least a portion of the channel 1665. In someembodiments, the energy source 1610 is configured to emit energy towardsonly specific targeted portions of the channel 1665. In someembodiments, this can be achieved through the use of the spatial energymodulating element or mask 1630. For example, the non-emitting portions1650 of the spatial energy modulating element 1630 may inhibit orprevent energy from being directed to one or more locations on thechannel 1665 (e.g., a location of a biological component). The emittingportions 1640 may allow energy emitted 1620 by the energy source 1610 tocontact targeted locations on the channel 1665. In some embodiments, themask 1630 comprising the emitting portions 1640 and the non-emittingportions 1650 may comprise physical components (e.g., an opaquematerial, a thermal shield, an electromagnetic shield, etc.). In otherembodiments, the mask 1630, the emitting portions 1640, the non-emittingportions 1650, or some combination thereof, may comprise digitalcomponents (e.g., a computer code or digital system to operateelectrodes, etc.). For example, a digital or a virtual mask may renderone or more electrodes, or arrays of electrodes, to producespatially-modulated energy (e.g., light, electrical current, etc.) toform the patterned polymer matrix 1660.

With continued reference to FIG. 16A, in some cases, the polymer matrix1660, or at least a portion of polymer matrix 1660, may surround, orsubstantially surround, the biological component 1670. In someembodiments, the polymer matrix 1660 may, by itself or in conjunctionwith the channel and other surfaces, may encapsulate the biologicalcomponent 1670.

In some embodiments, any of the surfaces of the channel or analysischannel may be configured to carry a functional group, as describedherein. In certain embodiments, the system may further comprise adetector for detecting (or identifying) the biological component, theone or more barcodes, or a combination thereof, as described herein. Invarious embodiments, the system may further comprise a platform or astage that is configured to hold the fluidic device. In someembodiments, the system may further comprise a sequencing device. Insome cases, the system may further comprise a spatial energy modulatingelement to selectively supply the energy to the fluidic device, asdescribed herein.

FIG. 16B shows a top view of a channel as provided herein. Emittedenergy from an energy source may be modulated spatially as describedherein to form a polymer matrix 1660. In some instances, the polymermatrix 1660 may encapsulate the biological component 1670. In certaininstances, the biological component may be encapsulated by the walls ofthe channel in and/or with a polymer matrix 1660.

FIG. 17A-17E illustrates an example workflow of applying energy to anyof the fluidic devices described herein to form a polymer matrix. FIG.17A shows a portion of a channel 1730 of a fluidic device, a firstbiological component 1720, a second biological component 1725, an energysource (e.g., a light source) 1700, and an energy modulating element(e.g., a mask or localized mask) 1710. In some embodiments, the energysource 1700 may be positioned adjacent to or near the channel 1730 suchthat energy sufficient to trigger hydrogel polymerization may bedirected from the energy source 1700 to at least a portion of thechannel 1730. In some embodiments, the spatial energy modulating element1710 may be used to selectively allow the energy from the energy source1700 to communicate with at least a portion of the fluidic device. Abiological sample or a biological component 1720, 1725 may be presentwithin the channel 1730. FIG. 17B shows a first step of hydrogelformation at a first location (e.g., adjacent to the first biologicalcomponent 1720). The energy source 1700 may emit energy 1750 sufficientto form a polymer matrix 1760 within the channel 1730 adjacent to thefirst biological component 1720.

In some embodiments, the energy may be guided by or passed through aspatial energy modulating element 1710, as described herein. In someexamples, a polymer matrix may form at locations in the channelcorresponding to emitting portions of the spatial energy modulatingelement 1710. FIG. 17C shows a second step of hydrogel formation in achannel 1730 where the relative positions of the energy source 1700 andthe channel 1730 may be changed. In some embodiments, the fluidic devicemay be placed on a movable stage. In certain embodiments, the energysource 1700 may be movable or coupled to a movable stage. In variousembodiments, the mask 1710 may be movable or coupled to a movable stage.In some embodiments, some combination of the energy source 1700, themask 1710, and the fluidic device may be configured to be movable withrespect to one another. In some embodiments, the spatial energymodulating element 1710 may be a virtual spatial energy modulatingelement, and the position of the emitting areas may be changeddigitally.

FIG. 17D shows a third step, where energy from the energy source 1700passing through the energy modulating element (or mask) 1710 may form apolymer matrix at a second location (e.g., adjacent to a secondbiological component 1765). In some embodiments, the energy source 1700,the mask 1710, and the fluidic device may be moved with respect to oneanother to form different hydrogel patterns comprising a plurality ofhydrogels matrices (e.g., hydrogel matrix 1765, 1766, 1767, and 1768)within the channel 1730 using energy from the energy source, asdescribed herein (FIG. 17E).

Methods of Analyzing Biological Components

Also provided herein are methods for analyzing biological components.The method may comprise introducing a biological component into afluidic device and forming a polymer matrix on or adjacent to thebiological component. The method may further include coupling thebiological component to one or more capture elements disposed on asurface (e.g., a first surface) of the fluidic device to yield a coupledbiological component. Accordingly, a polymer matrix may be formed on oradjacent to the coupled biological component.

FIG. 5 is a flow chart of an example of a method of analyzing abiological component using a system comprising a fluidic device asdisclosed herein. The method 500 may comprise providing and/orintroduced a biological sample 510 into the fluidic device, which maycomprise, or may be suspected to comprise, a biological component. In acapturing step 520, a capture element in the fluidic device may captureand/or couple the biological component, e.g., such that the biologicalcomponent is immobilized. In some cases, the immobilizing of thebiological component may be performed by using a repelling surfacecoating to contain a biological component within a portion of thesurface that may not have a repelling surface, as described herein. Insome cases, the capturing step may not be used, and the biologicalcomponents may be distributed over the surface randomly prior to step530. In step 530, a spatial energy modulating element (e.g., a mask) maybe applied selectively to provide energy from an energy source to thefluidic device to form one or more polymer matrices. In someembodiments, the mask may be configured to selectively supply energyadjacent to a biological component to form a polymer matrix adjacent tothe biological component. In certain embodiments, the mask may beconfigured to selectively supply energy on a biological component toform a polymer matrix that encapsulates at least a portion of thebiological component. The mask may be applied based at least in part ona location of the biological component. The method 500 may furthercomprise forming or generating a predefined or predetermined pattern ofcapture sites/elements on one or more surfaces (e.g., a first surface, asecond surface, a third surface, etc.) of the fluidic device before step510. In some cases, the predefined or predetermined pattern of capturesites/elements may be generated on the fluidic device prior to method500. In some embodiments, each capture site may be configured to carryone or more capturing element, as described herein.

In some embodiments, a detector, as provided herein, may be used todetect a location or position of a biological component. A mask may thenbe generated based at least in part on the detected location or positionof the biological component. In step 540, the generated mask may be usedin combination with an energy source to selectively apply energy to thefluidic device and/or the biological sample introduced into the fluidicdevice in step 510. In certain embodiments, the mask may be aphotolithographic mask or another suitable mask. In step 550, a polymermatrix may be formed by applying energy sufficient to polymerize thepolymer precursors in the fluidic device and/or the biological sampleintroduced into the fluidic device in 510 (e.g., from the energysource). The energy may comprise electrochemical energy, electromagneticenergy, thermal energy, microwave energy, or any other suitable energy.In some embodiments, the energy may be light energy, as discussedherein.

In certain embodiments, the polymer matrix may be formed adjacent to abiological component. In some embodiments, the polymer matrix may beformed on a biological component (e.g., to encapsulate the biologicalcomponent). The polymer matrix may be formed in between two biologicalcomponents to prevent contact (e.g., physical contact) between the twobiological components. In various embodiments, at least a portion of thebiological component may be surrounded by the polymer matrix. In someembodiments, one or more biological components in the biological samplemay be surrounded by a polymer matrix such that two biologicalcomponents may be separated from one another by a polymer matrix (e.g.,one or more polymer matrix walls). The biological component may beencapsulated by the polymer matrix. In certain embodiments, the polymermatrix may comprise a hydrogel. In various embodiments, the polymermatrix may form a compartment around the biological component (e.g., bysurrounding a biological component) to form an analysis chamber.

As described elsewhere herein, one or more assays may be conducted orperformed on the biological component in the analysis chamber. One ormore biological components in the biological sample may be captured andan analysis chamber may be formed adjacent to and/or surrounding each ofthe captured biological components. The order of the steps or actions ofthe methods described in connection with the embodiments disclosed maybe varied. Thus, any order in the drawings or detailed description isfor illustrative purposes only and is not meant to imply a requiredorder, unless otherwise specified.

In some embodiments, one or more functional assays may be conducted orperformed on the biological component in the polymer matrix. In someembodiments, the functional assays will be used to assess cellviability, cell morphology, cell secretions, cell responses,intercellular interactions, or any combination thereof. In someembodiments, a colorimetric assay or fluorescent assay may be conductedor performed. In some embodiments, a functional assay is performed usingbright-field phase contrast or fluorescent imaging of the analyte. Insome embodiments, a cell may be lysed, and a functional assay will beperformed on a component thereof. Because one or more individualcomponents can be localized within a fluidic device (e.g., encapsulated)and the localized components be exposed to one or more reagents and/orwashing solutions during and/or in between analyses, multiple assays canbe performed within the compartments (e.g., simultaneously,substantially simultaneously, serially, etc.). Different assays may beperformed in different locations of the fluidic device, for example, totest effects of different treatment conditions.

In some embodiments, one or more omics assays may be performed tocharacterize and quantify the biological component or a componentthereof. The omics assay may be a proteomic, transcriptomic, genomic, orepigenomic assay, or any combination thereof. In some embodiments, theone or more omics assays is a multi-omic assay.

Also provided herein are methods for obtaining a transcriptome of abiological component. The method may comprise coupling the biologicalcomponent to a capture element disposed in a fluidic device to yield acoupled biological component. The method may include forming a polymermatrix on or adjacent to the coupled biological component to form ananalysis chamber. The method may further include conducting orperforming one or more reactions in the analysis chamber to obtain thetranscriptome of the biological component. The biological component mayremain in the analysis chamber during performance of the one or morereactions. In some embodiments, the biological component may not becoupled to a capture element disposed in a fluidic device. For example,the biological component can be localized by forming a polymer matrix onor adjacent to the biological component without coupling the biologicaldevice to the capture element. The transcriptome may be captured bytarget capture probes on the surface. The capture probe can include abarcode which decodes the location of the biological component anddifferentiates the source biological component. Further biochemicalprocessing can be performed on the captured transcriptome of thebiological component in order to convert it to DNA and determine itssequence using a form of sequencing or hybridization.

The method may further comprise directing energy from an energy sourceto at least a portion of the fluidic device to form the polymer matrixon or adjacent to the biological component to form the compartment oranalysis chamber. The energy may be directed to the fluidic deviceselectively to form analysis chambers at predefined locations in thefluidic device. The selective directing of the energy may be performedusing a spatial energy modulating element (e.g., a mask as describedherein). In some embodiments, the method may further comprise detectinga biological component using a detector. Information from the detectedbiological component (e.g., a location of the biological component inthe fluidic device) may be used to form or generate the spatial energymodulating element. For example, the spatial energy modulating elementmay inhibit or prevent energy from being directed to a location adjacentto the biological component. In some embodiments, the spatial energymodulating element may selectively direct energy to a location adjacentto the biological component. In some embodiments, the spatial energymodulating element may selectively direct energy on the biologicalcomponent.

In certain cases, the method may comprise directing energy from anenergy source to a predetermined portion of the fluidic device to formpolymer matrix structures at predetermined locations in the fluidicdevice or in predetermined patterns in the fluidic device. In someembodiments, a detector (e.g., to detect the locations of one or morebiological components) may be used. In various embodiments, a detectormay not be used. A certain number of the polymer matrix structures maybe formed or generated on or adjacent to a biological component. Statedanother way, there may be a sufficient number of biological componentsin the fluidic device such that it is not necessary to first determinethe locations of the biological components before forming the polymermatrix structures.

The spatial energy modulating element may comprise a physical photomask,a virtual photomask, a physical electrode distribution pattern, avirtual electrode distribution pattern, a photolithographic mask, a DMDsystem, or any other suitable mask. A physical or virtual photomask may,for example, prevent energy from being directed to a portion of thefluidic device while allowing energy to be directed to another portionof the fluidic device. An electrode distribution pattern may compriseelectrically activating one or more electrodes (e.g., an array ofelectrodes) to allow energy to be directed to a portion of the fluidicdevice. An electrode may be rendered “off” or incapable of producingenergy in a location where a polymer matrix may not be formed. Anelectrode may be rendered “on” at a location where a polymer matrix maybe formed.

In some cases, the biological component may comprise a cell or atranscriptome thereof. A cell may comprise a eukaryotic cell, aprokaryotic cell, a fungal cell, an algal cell, a protozoan, a plantcell, an animal cell (e.g., a human cell), or any other suitable cell.The one or more reactions performed may comprise RNA sequencing. In someembodiments, the one or more reactions performed may comprise analysisof a transcriptome (e.g., under different or varying conditions). Forexample, one or more reactions may be performed to analyze the effect oreffects of temperature, a small molecule, a toxin, a cell-cellinteraction, an infection, etc. on a transcriptome. In some embodiments,the analysis of the transcriptome may provide a gene expression profilefor a cell or a combination of cells. In certain embodiments, the one ormore reactions performed may comprise a hybridization-based readout ofthe gene expression. The transcriptome may comprise messenger RNA(mRNA), long non-coding RNA (lncRNA), mitochondrial RNA, or total RNA.In some embodiments, other types of RNA (e.g., ribosomal RNA (rRNA)) maybe analyzed and or sequenced. The RNA may comprise at least 50nucleotides (nt), 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700nt, 800 nt, 900 nt, 1,000 nt, 10,000 nt, or more nucleotides. Thebiological component may comprise any number of nucleotides in betweenany two numbers mentioned herein. The RNA may be a double-stranded RNA(dsRNA) molecule or a single-stranded RNA (ssRNA) molecule.

Also provided herein are methods for analyzing two or more biologicalcomponents. The method may comprise introducing a first biologicalcomponent and a second biological component into a fluidic device. Themethod may include forming a polymer matrix on or adjacent to the firstbiological component to form a first analysis chamber. The method mayfurther include forming a polymer matrix on or adjacent to the secondbiological component to form a second analysis chamber. The firstanalysis chamber may be adjacent to the second analysis chamber. Themethod may further comprise, analyzing one or more features of the firstbiological component and/or the second biological component.

In some embodiments, one or more features of the first biologicalcomponent and/or the second biological component may comprise a responseto an analyte, a response to a pharmaceutical agent, a response to anantimicrobial agent, production of a target compound by a cell or acommunity of cells, production of a target molecule, production of anucleic acid, production of a protein, or any other suitable response.The one or more features may comprise at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 25, 50, 100, or more features of the first biologicalcomponent. The one or more features may comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 25, 50, 100, or more features of the secondbiological component. In some cases, a feature of the first biologicalcomponent may be compared to the same feature of the second biologicalcomponent. In various embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25,50, 100, or more features of the first biological component and/or thesecond biological component may be analyzed or monitored. In certainembodiments, a feature of the first biological component may be comparedto a different feature of the second biological component. For example,a first feature in the first biological component may comprise aresponse to an analyte or a pharmaceutical agent causing the firstbiological component to generate a compound or molecule (e.g., anantibody, a protein, an enzyme, etc.). A second feature in the secondbiological component may then comprise a response of the secondbiological component to the molecule or compound generated by the firstbiological component.

In some cases, a first feature and a second feature in the firstbiological component or the second biological component may be analyzed.For example, the first feature and the second feature may be analyzed inthe first biological component in a response to a compound or a moleculegenerated by the second biological component. This may be used toelucidate one or more interactions between the first biologicalcomponent and the second biological component.

The interaction may comprise a communication (e.g., a biologicalcommunication) between the first and the second biological components.In some cases, the interaction may comprise a biochemical communicationbetween the first and the second biological components. In someembodiments, the biological communication may comprise a moleculeincluding a protein, a nucleic acid, a cytokine, a chemokine, acombination thereof, or any other suitable molecule. The molecule may begenerated by the first biological component or by the second biologicalcomponent. In some cases, more than two biological components may belocalized in analysis chambers formed adjacent to one another to analyzethree or more features in three or more biological components. In someembodiments, this may be used to investigate interaction(s) betweenthree or more biological components. In various embodiments,interactions between 2, 3, 4, 5, 6, 7, 10, 15, 25, 50, 100, or morebiological components may be investigated.

In some cases, the first and the second biological components mayinclude different cell types (e.g., a first cell type and a second celltype). In certain embodiments, the first biological component and thesecond biological component may include similar cell types, for example,cell types with varying genotypes or phenotypes. A response (e.g., alevel of a response) to an analyte or pharmaceutical compound can becompared between two different cell types. In some cases, an amount of atarget compound or an amount of molecule production between twodifferent cell types can be analyzed and/or compared. In someembodiments, the first biological component and the second biologicalcomponent may remain localized within the first analysis chamber and thesecond analysis chamber, respectively, during the analysis of thefeatures. Such analysis or experiments can be scaled up to assess oranalyze 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 100 or more biologicalcomponents (e.g., in parallel).

Also provided herein are methods for identifying a nucleic acidmolecule. This method may comprise the capture of a biologicalcomponent. The biological component may then be encapsulated by ahydrogel, which may form an analysis chamber. In some embodiments, thenucleic acid molecule can be extracted and released within the analysischamber. This nucleic acid may be sequenced by next-generationsequencing, sequencing may be performed through short-read sequencing,nanopore sequencing, sequencing by synthesis, sequencing by in-situhybridization, or any optical readout using, for example, a microscope.Other suitable methods of sequencing are also within the scope of thisdisclosure. The method may further comprise detecting the nucleic acidmolecule in an absence of nucleic acid amplification.

In some embodiments, a biological component comprising a cell may beintroduced into the fluidic device. A polymer matrix may then be formedon or adjacent to the biological component to form a compartment oranalysis chamber. The analysis chamber may be formed by selectivelydirecting energy from an energy source to the fluidic device, asdescribed herein. In some embodiments, the analysis chamber may bedeconstructed by degrading the polymer matrix “on demand.” Degrading thepolymer matrix on demand may comprise selectively directing energy tothe analysis chamber, using an enzyme to digest or depolymerize thepolymer matrix, or any other suitable method for degrading a polymermatrix.

A cell may be lysed to release a biological component (e.g., DNA orRNA). In certain embodiments, a biological component is released from acell upon interaction with a reagent. In some embodiments, the reagentis an organic or inorganic molecule. In some embodiments, the organic orinorganic molecule is a pharmaceutical compound or detergent. In someembodiments, the reagent is a protein. In some embodiments, the reagentis a DNA aptamer. In some embodiments, the reagent is a bead carryingbiomolecules. In some embodiments, the reagent is a biological species.In some embodiments, the biological species is a virus or cell.

In some embodiments, a biological component is released from the cellupon exposure to an energy source. In some embodiments, the energysource is UV light for lysing cells. In some embodiments, the energysource is visible light for lysing cells. In some embodiments, the UVlight is used to activate a photoactivated detergent and lyse the cell.In some embodiments, the visible light is used to activate aphotoactivated detergent and lyse the cell.

As described herein, the polymer matrix may have a pore size or anaverage pore size that may not allow the nucleic acid molecules to passor traverse through the polymer matrix. In some cases, the analysischamber may be used for sequencing library preparation and/or nucleicacid sequencing. The sequencing can be next-generation sequencing. Thesequencing can be short-read sequencing, nanopore sequencing, sequencingby synthesis, sequencing by in-situ hybridization, or an optical readoutusing, for example, a microscope. The one or more nucleic acid moleculesin the analysis chamber may undergo nucleic acid sequencing reaction(s)comprising, for example, DNA sequencing or RNA sequencing. A nucleicacid library may be constructed or generated. The polymer matrix mayallow reagents to pass through, as described herein. The reagents maycomprise primers, adapters, enzymes, and other reagents used for nucleicacid sequencing reactions.

In certain embodiments, a nucleic acid may comprise a DNA molecule or anRNA molecule. The DNA may be at least 100 bp long. In some embodiments,the DNA may include at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 600 bp, 700 bp, 800 bp, 900 bp, 1,000 bp, 10 kilo base pairs (kbp),100 kbp, 1 mega base pairs (Mbp), 100 Mbp, 1 giga base pairs (Gbp), 10Gbp, 100 Gbp, or more base pairs. The biological component may comprisea DNA molecule that comprises any number of base pairs in between thementioned numbers herein. For example, the DNA may comprise from 50 bpto 1,000 bp, 300 bp to 10 kbp, or 1,000 bp to 10 Gbp. The RNA may bedsRNA. The dsRNA may comprise at least 50 bp, 100 bp, 200 bp, 300 bp,400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1,000 bp, 10 kbp, or 100kbp. The biological component may comprise a dsRNA molecule thatcomprises any number of base pairs in between the mentioned numbersherein. For example, the dsRNA may comprise from 50 bp to 1,000 bp, 300bp to 10 kbp, or 1,000 bp to 100 kbp. The RNA may be ssRNA. The ssRNAmay comprise from 50 nt to 100,000 nt. The ssRNA may comprise from 50 ntto 100 nt, 50 nt to 1,000 nt, 50 nt to 10,000 nt, 50 nt to 100,000 nt,100 nt to 1,000 nt, 10 nt to 100 nt, 10 nt to 1,000 nt, or 100 nt to1,000 nt. In some cases, the ssRNA may be less than 50 nucleotides long.The ssRNA may be more than 100,000 nucleotides long.

In some embodiments, the sequencing library construction may comprisebarcoding. The barcoding may be unique in each analysis chamber toassociate a nucleic acid molecule to an analysis chamber or a biologicalcomponent (e.g., a cell). In some embodiments, a polymerase chainreaction (PCR) may be used for the sequencing library construction. Insome cases, because the nucleic acid material may be retained within ananalysis chamber and may not be diluted or lost, PCR may not be usedother than for the sequencing library construction. In some embodiments,the sequencing library construction may comprise adapter ligation. Insome embodiments, all the steps required for sequencing a nucleic acidmolecule may be performed while the biological component and/or thenucleic acids are localized in the analysis chamber. In someembodiments, library preparation comprises transposase-assistedtagmentation of the nucleic acid molecules (e.g., by using transposonsto cleave and label genomic DNA).

Also provided herein are methods for processing a cell to determine atranscriptome of the cell. In some embodiments, this method does notcomprise nucleic acid amplification. In other embodiments, this methodmay comprise nucleic acid amplification. The method may includeprocessing the cell to determine an epigenome of the cell. In someembodiments, a biological component comprising a cell may be introducedinto a fluidic device as provided herein. A polymer matrix may be formedon or adjacent to the biological component to form an analysis chamber(e.g., around the biological component or that encapsulates thebiological component). The analysis chamber may at least partially orcompletely surround the cell. The analysis chamber may be formed byselectively directing energy from an energy source to the fluidicdevice, as described herein. In some embodiments, the analysis chambermay be deconstructed or removed by degrading the polymer matrix. Thepolymer matrix may be degraded “on demand,” as provided herein.

The biological component or products thereof may be analyzed within theanalysis chamber. In some cases, the biological component or productsthereof may be eluted and transferred to another device for analysis.For example, nucleic acid material or protein products from a biologicalcomponent (e.g., a cell, bacteria, virus, etc.) may be extracted and/orprocessed (e.g., tagged, barcoded, etc.) in an analysis chamber. Thenucleic acid material or protein products may then be sequenced withinthe analysis chambers of the fluidic device described herein. In somecases, the nucleic acid material or protein products may be eluted andtransferred to another device (e.g., a sequencing flow cell) to besequenced (e.g., using a sequencing device).

The cell may comprise a mammalian cell (e.g., a human cell), a fungalcell, a bacterial cell, an algal cell, a protozoan, a plant cell, atumor spheroid, or a combination thereof. In some embodiments, a genome,transcriptome, proteome, epigenome, methylome, secretome, or metabolomeof the cell may be extracted, for example, by lysing the cell. In somecases, one or more proteins produced and/or released by the cell can beanalyzed without lysing the cell. In some cases, the genome,transcriptome, proteome, epigenome, methylome, secretome, and/ormetabolome of the cell can be studied, analyzed, and/or sequenced (asappropriate) while the cell and/or the genome, transcriptome, proteome,epigenome, methylome, secretome, or metabolome of the cell may remainwithin the analysis chamber, or may remain substantially within theanalysis chamber. In some embodiments, the nucleic acid in the cell mayremain within the analysis chamber. Accordingly, nucleic acidamplification may be avoided or obviated. In some cases, one or morecells may be processed within an analysis chamber to identify and studya genome, transcriptome, proteome, epigenome, methylome, secretome,and/or metabolome in the one or more cells.

A first surface and/or a second surface of the fluidic device (similarto the surfaces 101, 102 of FIG. 1) may comprise detecting elements. Thedetecting elements may comprise one or more reagents or biochemicalsensors. In some cases, the reagents to study the genome, transcriptome,proteome, epigenome, methylome, secretome, and/or metabolome of the cellmay be introduced into the fluidic device. The polymer matrix maycomprise pores allowing the reagents to pass through, but the pores maynot allow nucleic acids or proteins (e.g., of the cell) to cross thepolymer matrix.

Also provided herein are methods for identifying a plurality of nucleicacid molecules of a plurality of cells without barcoding individualnucleic acid molecules of the plurality of nucleic acid molecules. Themethod may comprise sequencing the plurality of nucleic acid molecules.In some embodiments, a plurality of biological components may beintroduced into the fluidic device. A polymer matrix may be formed on oradjacent to a biological component from the plurality of biologicalcomponents to form an analysis chamber that at least partially orcompletely surrounds or encapsulates the biological component. Theanalysis chamber may be formed by selectively directing energy from anenergy source to the fluidic device, as described herein. In someembodiments, the analysis chamber may be deconstructed by degrading thepolymer matrix “on demand,” as described herein. The plurality ofnucleic acid molecules of the biological component (e.g., a cell, abacteria, a virus, etc.) may be extracted in the analysis chamber formedsurrounding the biological component. The nucleic acid molecules maythen be sequenced within the analysis chamber. A readout of thesequencing may be performed within the analysis chamber. Therefore, aneed for a barcode to associate a nucleic acid molecule and thebiological component may be avoided.

The plurality of nucleic acid molecules may be extracted from theplurality of cells. In some cases, a nucleic acid molecule of a cell maybe extracted from the cell by lysing the cell within the analysischamber (e.g., the compartment or chamber surrounding the cell). Theanalysis chamber may contain and/or localize the nucleic acid from thecell within the analysis chamber. Therefore, the plurality of nucleicacid molecules from the plurality of cells may be localized inindividual and/or separate analysis chambers. In some embodiments, thesequencing process (e.g., nucleic acid library construction, sequencing,etc.) may be performed on the plurality of nucleic acid molecules thatmay be isolated or localized in separate analysis chambers. Barcoding todistinguish between a cell generating a nucleic acid molecule in theplurality of the nucleic acid molecules may therefore be avoided orobviated.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 15 shows a computer system1501 that may be programmed or otherwise configured to perform methodsdescribed herein. The computer system 1501 can regulate various aspectsof the present disclosure, such as, for example, identifying abiological component, detecting a barcode, generating a spatialmodulating element (e.g., a mask), providing energy from an energysource, or detecting or measuring a local parameter using a sensor. Thedetector may be a camera (e.g., a fluorescent camera), such as a chargedcoupled device (CCD) camera capable of collecting optical signals andposition information from a plurality of sources distributed over aplanar region. The computer system 1501 can be an electronic device of auser or a computer system that may be remotely located with respect tothe electronic device. The electronic device can be a mobile electronicdevice.

The computer system 1501 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1505, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1501 also includes memory or memorylocation 1510 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1515 (e.g., hard disk), communicationinterface 1520 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1525, such as cache, othermemory, data storage and/or electronic display adapters. The memory1510, storage unit 1515, interface 1520 and peripheral devices 1525 arein communication with the CPU 1505 through a communication bus (solidlines), such as a motherboard. The storage unit 1515 can be a datastorage unit (or data repository) for storing data. The computer system1501 can be operatively coupled to a computer network (“network”) 1530with the aid of the communication interface 1520. The network 1530 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that may be in communication with the Internet. The network1530 in some cases may be a telecommunication and/or data network. Thenetwork 1530 can include one or more computer servers, which can enabledistributed computing, such as cloud computing. The network 1530, insome cases with the aid of the computer system 1501, can implement apeer-to-peer network, which may enable devices coupled to the computersystem 1501 to behave as a client or a server.

The CPU 1505 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1510. The instructionscan be directed to the CPU 1505, which can subsequently program orotherwise configure the CPU 1505 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1505 can includefetch, decode, execute, and writeback.

The CPU 1505 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1501 can be included in thecircuit. In some cases, the circuit may be an application specificintegrated circuit (ASIC).

The storage unit 1515 can store files, such as drivers, libraries, andsaved programs. The storage unit 1515 can store user data, e.g., userpreferences and user programs. The computer system 1501 in some casescan include one or more additional data storage units that are externalto the computer system 1501, such as located on a remote server that maybe in communication with the computer system 1501 through an intranet orthe Internet.

The computer system 1501 can communicate with one or more remotecomputer systems through the network 1530. For instance, the computersystem 1501 can communicate with a remote computer system of a user(e.g., a laptop, a personal computer, a tablet, or a mobile phone).Examples of remote computer systems include personal computers (e.g.,portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® GalaxyTab), telephones, Smart phones (e.g., Apple® iPhone, Android-enableddevice, Blackberry®), or personal digital assistants. The user canaccess the computer system 1501 via the network 1530.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1501, such as, for example, on thememory 1510 or electronic storage unit 1515. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1505. In some cases, thecode can be retrieved from the storage unit 1515 and stored on thememory 1510 for ready access by the processor 1505. In some situations,the electronic storage unit 1515 can be precluded, andmachine-executable instructions are stored on memory 1510.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1501, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that may be carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1501 can include or be in communication with anelectronic display 1535 that comprises a user interface (UI) 1540 forproviding, for example, an image of a biological component, a barcode, asignal or measurement of a local parameter. Examples of UI's include,without limitation, a graphical user interface (GUI) and web-based userinterface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1505. Thealgorithm can, for example, identify a biological component, detect abarcode, generate a spatial modulating element (e.g., a mask), provideenergy from an energy source, detect or measure a local parameter usinga sensor, etc.

EMBODIMENTS

Embodiment 1. A system, comprising: a fluidic device containing one ormore biological components and one or more polymer precursors; and atleast one energy source in communication with said fluidic device,wherein said at least one energy source supplies energy to said fluidicdevice to cause said one or more polymer precursors to form at least onepolymer matrix on or adjacent to said biological component.

Embodiment 2. The system of Embodiment 1, wherein said fluidic devicecomprises a channel disposed therethrough, and wherein a first surfaceis disposed along a portion of said channel, and a second surface isdisposed opposite of said first surface.

Embodiment 3. The system of Embodiment 1 or Embodiment [00325], whereinsaid fluidic device comprises a chamber disposed therein, wherein afirst surface is disposed along a portion of said chamber, and a secondsurface is disposed opposite of said first surface.

Embodiment 4. The system of Embodiment [00325] or Embodiment [00326],wherein said first surface is a lower surface, and wherein said secondsurface is an upper surface.

Embodiment 5. The system of Embodiment 1, wherein said fluidic devicefurther comprises one or more capture elements immobilizing at least oneof said one or more biological components at a location adjacent to saidfirst surface forming an immobilized biological component.

Embodiment 6. The system of Embodiment 1, wherein said first surface isdisposed adjacent to said energy source, and wherein said energy sourceis an array of electrodes.

Embodiment 7. The system of Embodiment [00329], wherein said array ofelectrodes supply electrochemical energy to said one or more polymerprecursors to form an array of polymer matrices.

Embodiment 8. The system of Embodiment 1, wherein at least two of saidone or more polymer precursors are coupled to said first surface forminga pattern on said first surface.

Embodiment 9. The system of Embodiment [00331], wherein said at leastone polymer matrix is formed on or adjacent to said pattern.

Embodiment 10. The system of Embodiment [00328], wherein said at leastone polymer matrix is coupled to said first surface.

Embodiment 11. The system of Embodiment [00333], wherein said at leastone polymer matrix extends from said first surface to said secondsurface such that said at least one polymer matrix surrounds at least aportion of said immobilized biological component.

Embodiment 12. The system of any one of Embodiments 1 to [00334],wherein said at least one energy source is in at least one of opticalcommunication, electrochemical communication, electromagneticcommunication, thermal communication, or microwave communication withsaid fluidic device.

Embodiment 13. The system of any one of Embodiments 1 to [00334],wherein said at least one energy source comprises a light generatingdevice, a heat generating device, an electrochemical generating device,an electrode, or a microwave device.

Embodiment 14. The system of any one of Embodiments 1 to [00336],further comprising a photolithographic device or a digital micromirrordevice (DMD) configured to control a spatial distribution of said energyfrom said energy source.

Embodiment 15. The system of any one of Embodiments [00328] to [00337],wherein said one or more capture elements comprise a physical trap, ageometric trap, a well, an electrochemical trap, a chemical affinitytrap, one or more magnetic particles, an electrophoretic trap, adielectrophoretic trap, or a combination thereof.

Embodiment 16. The system of Embodiment [00338], wherein said chemicalaffinity trap comprises streptavidin, an antibody, or a combinationthereof.

Embodiment 17. The system of Embodiment [00338], wherein the physicaltrap comprises a polymer matrix.

Embodiment 18. The system of Embodiment [00340], wherein the polymermatrix comprises a hydrogel.

Embodiment 19. The system of Embodiment [00338], wherein saidelectrochemical trap comprises a gold electrode, a platinum electrode,or an indium tin oxide (ITO) electrode.

Embodiment 20. The system of any one of Embodiments [00328] to [00342],wherein said one or more capture elements are disposed in a pattern onsaid first surface or said second surface.

Embodiment 21. The system of any one of Embodiments [00328] to [00343],wherein said one or more capture elements comprises a well, wherein saidwell is from 1 μm (micrometer) to 50 μm in diameter, and wherein saidwell is from 0.1 μm to 100 μm in depth.

Embodiment 22. The system of any one of Embodiments [00328] to [00344],wherein said one or more biological components is a plurality ofbiological components, and wherein said plurality of biologicalcomponents is coupled to said one or more capture elements.

Embodiment 23. The system of any one of Embodiments 1 to [00345],wherein said fluidic device is a microfluidic device or a nanofluidicdevice.

Embodiment 24. The system of any one of Embodiments 1 to [00345],wherein said fluidic device is used for nucleic acid sequencing.

Embodiment 25. The system of Embodiment [00347], wherein said nucleicacid sequencing comprises next-generation sequencing, short-readsequencing, nanopore sequencing, sequencing by synthesis, sequencing byin-situ hybridization, or an optical readout.

Embodiment 26. The system of any one of Embodiments 1 to [00348],wherein said one or more biological components comprise a cell, a celllysate, a nucleic acid, a microbiome, a protein, a mixture of cells, aspatially-linked biological component, or a metabolite.

Embodiment 27. The system of Embodiment [00349], wherein said mixture ofcells comprises a first cell type and a second cell type, and whereinsaid first cell type is different than said second cell type.

Embodiment 28. The system of Embodiment [00349], wherein said cell is ananimal cell, a plant cell, a fungal cell, or a bacterial cell.

Embodiment 29. The system of Embodiment [00351], wherein said animalcell is a human cell.

Embodiment 30. The system of any one of Embodiments 1 to [00351],wherein said one or more biological components comprise a tumor spheroidor a spatially linked biological sample.

Embodiment 31. The system of any one of Embodiments [00349] to [00353],wherein said nucleic acid is DNA of 100 base pairs or greater or RNA of50 bases or greater.

Embodiment 32. The system of any one of Embodiments [00349] to [00354],wherein said cell lysate comprises DNA from 50 bp (base pairs) to 100Gbp (giga base pairs) or RNA from 50 bp to 100 kbp (kilo base pairs).

Embodiment 33. The system of any one of Embodiments 1 to [00355],wherein said at least one polymer matrix comprises a hydrogel.

Embodiment 34. The system of any one of Embodiments 1 to [00356],wherein said fluidic device further comprises one or more polymerprecursors.

Embodiment 35. The system of Embodiment [00356], wherein said one ormore polymer precursors comprise hydrogel precursors.

Embodiment 36. The system of any one of Embodiments [00328] to [00358],wherein said at least one polymer matrix inhibits passage of saidimmobilized biological component.

Embodiment 37. The system of any one of Embodiments 1 to [00359],wherein said at least one polymer matrix forms a cylinder shell or apolygon shell comprising an inner space and a polymer matrix wall.

Embodiment 38. The system of Embodiment [00360], wherein said innerspace has an inner diameter from 1 μm to 500 μm.

Embodiment 39. The system of Embodiment [00360] or Embodiment [00361],wherein said polymer matrix wall has a thickness of at least 1 μm(micrometer).

Embodiment 40. The system of Embodiment [00362], wherein said at leastone polymer matrix wall is a hydrogel wall.

Embodiment 41. The system of any one of Embodiments 1 to [00363],wherein said at least one polymer matrix is degradable.

Embodiment 42. The system of Embodiment [00364], wherein saiddegradation of said at least one polymer matrix is “on demand.”

Embodiment 43. The system of Embodiment [00364] or Embodiment [00365],wherein said at least one polymer matrix is degradable by at least oneof: (i) contacting said at least one polymer matrix with a cleavingreagent; (ii) heating said at least one polymer matrix to at least 90°C.; or (iii) exposing said at least one polymer matrix to a wavelengthof light that cleaves a photo-cleavable crosslinker that crosslinks saidpolymer of said at least one polymer matrix.

Embodiment 44. The system of Embodiment [00366], wherein said at leastone polymer matrix comprises a hydrogel, and wherein said cleavingreagent degrades said hydrogel.

Embodiment 45. The system of Embodiment [00366], wherein said cleavingreagent comprises a reducing agent, an oxidative agent, an enzyme, a pHbased cleaving reagent, or a combination thereof.

Embodiment 46. The system of Embodiment [00366], wherein said cleavingreagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine(TCEP), tris(3-hydroxy propyl)phosphine (THP), or a combination thereof.

Embodiment 47. The system of any one of Embodiments 1 to [00369],wherein said at least one polymer matrix allows passage of a reagent.

Embodiment 48. The system of any one of Embodiments 1 to [00370],wherein said at least one polymer matrix comprises pores, wherein anaverage size of said pores is modulated using a chemical reagent, byapplying heat, applying electricity, applying light, or a combinationthereof.

Embodiment 49. The system of Embodiment [00370] or Embodiment [00371],wherein said reagent comprises at least one of enzymes, chemicals,oligonucleotides, or primers having a size of less than 50 base pairs.

Embodiment 50. The system of any one of Embodiments [00370] to [00372],wherein said reagent comprises lysozyme, proteinase K, random hexamers,polymerase, transposase, ligase, catalyzing enzyme, deoxynucleotidetriphosphates, buffers, cell culture media, or divalent cations.

Embodiment 51. The system of any one of Embodiments 1 to [00373],wherein said at least one polymer matrix comprises pores that are sizedto allow diffusion of a reagent through said at least one polymer matrixbut are too small to allow DNA or RNA to traverse said pores.

Embodiment 52. The system of any one of Embodiments 1 to [00374],wherein said at least one polymer matrix comprises a hydrogel, andwherein said hydrogel comprises polyethylene glycol (PEG)-thiol,PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine, PEG,polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, bisacrylamide, diacrylate, diallylamine,triallylamine, divinyl sulfone, diethyleneglycol diallyl ether,ethyleneglycol diacrylate, polymethyleneglycol diacrylate,polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate,ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritoltetraacrylate, or combinations or mixtures thereof.

Embodiment 53. The system of Embodiment [00375], wherein said hydrogelcomprises an enzymatically degradable hydrogel, PEG-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/PPO.

Embodiment 54. The system of any one of Embodiments [00328] to [00376],wherein said first surface, said second surface, or both comprise one ormore barcodes.

Embodiment 55. The system of Embodiment [00377], wherein said one ormore barcodes comprise an identifier to identify a source of said one ormore biological components.

Embodiment 56. The system of Embodiment [00378], wherein said one ormore barcodes comprise an oligonucleotide.

Embodiment 57. The system of Embodiment [00378], wherein said sourcecomprises a specimen from which said one or more biological componentsare collected.

Embodiment 58. The system of Embodiment [00378], wherein said sourcecomprises a physiological or an anatomical source from which said one ormore biological components are collected.

Embodiment 59. The system of Embodiment [00381], wherein said anatomicalsource comprises an organ of a subject.

Embodiment 60. The system of Embodiment [00382], wherein said subject isa human.

Embodiment 61. The system of any one of Embodiments [00377] to [00383],wherein said one or more barcodes are configured to bind said one ormore biological components or a molecule made by said one or morebiological components.

Embodiment 62. The system of any one of Embodiments [00328] to [00384],wherein said first surface, said second surface, or both comprise one ormore compounds configured to bind said one or more biologicalcomponents.

Embodiment 63. The system of any one of Embodiments [00328] to [00385],wherein said first surface, said second surface, or both isfunctionalized with a surface polymer.

Embodiment 64. The system of Embodiment [00386], wherein said surfacepolymer is functionalized with an oligonucleotide.

Embodiment 65. The system of Embodiment [00386], wherein said surfacepolymer is functionalized with an antibody, a cytokine, a chemokine, aprotein, an antibody derivative, an antibody fragment, a carbohydrate, atoxin, an aptamer, or any combination thereof.

Embodiment 66. The system of any one of Embodiments 1 to [00388],wherein said polymer matrix is functionalized with an oligonucleotide,an antibody, a cytokine, a chemokine, a protein, an antibody derivative,an antibody fragment, a carbohydrate, a toxin, an aptamer, or anycombination thereof.

Embodiment 67. The system of Embodiment [00386], wherein said surfacepolymer comprises polyethylene glycol (PEG), a silane polymer,pyridinecarboxaldehyde (PCA), an acrylamide, an agarose, or acombination thereof.

Embodiment 68. The system of any one of Embodiments 1 to [00390],further comprising a detector for identifying said one or more of saidbiological components, said one or more barcodes, or a combinationthereof.

Embodiment 69. The system of Embodiment [00391], wherein said detectorcomprises a camera (fluorescent camera).

Embodiment 70. The system of any one of Embodiments 1 to [00392],further comprising a stage that holds said fluidic device.

Embodiment 71. The system of any one of Embodiments 1 to [00393],further comprising a sequencing device for obtaining sequencing data.

Embodiment 72. The system of Embodiment [00394], wherein said sequencingdata is generated using next-generation sequencing, short-readsequencing, nanopore sequencing, sequencing by synthesis, sequencing byin-situ hybridization, or an optical readout.

Embodiment 73. The system of any one of Embodiments 1 to 72, furthercomprising a spatial energy modulating element to selectively supplysaid energy to said fluidic device.

Embodiment 74. The system of Embodiment [00396], wherein said spatialenergy modulating element is generated using said detector identifyingsaid position of said at least one biological component.

Embodiment 75. The system of Embodiment [00396] or Embodiment [00397],wherein said spatial energy modulating element comprises a physicalphotomask, a virtual photomask, a physical electrode distributionpattern, or a virtual electrode distribution pattern.

Embodiment 76. The system of Embodiment [00396] or Embodiment [00397],wherein said spatial energy modulating element comprises aphotolithographic mask or a digital micromirror device (DMD) mask.

Embodiment 77. A method of analyzing a biological component, said methodcomprising: (a) introducing one or more biological components into afluidic device; (b) disposing a first portion of said one or morebiological components adjacent a first surface or a second surface ofsaid fluidic device; and (c) forming one or more polymer matricesadjacent a first portion of said first surface or said second surface tolocalize at least one of said one or more biological components to saidfirst portion.

Embodiment 78. The method of Embodiment [00400], further comprising: (a)agitating said one or more biological components within said fluidicdevice; (b) disposing a second portion of said one or more biologicalcomponents adjacent said first surface or said second surface of saidfluidic device; and (c) forming one or more polymer matrices adjacent asecond portion of said first surface or said second surface toimmobilize at least one of said one or more biological component of saidsecond portion.

Embodiment 79. The method of Embodiment [00400] or Embodiment [00401],further comprising identifying a position of at least one of said one ormore biological components such that at least one energy source suppliesenergy to said fluidic device to form said one or more polymer matriceson or adjacent said identified position.

Embodiment 80. A method of analyzing a biological component, comprising:(a) introducing said biological component into a fluidic device; (b)coupling said biological component to one or more capture elementsdisposed on a first surface or a second surface of said fluidic deviceto yield a coupled biological component; and (c) forming a polymermatrix on or adjacent to said coupled biological component.

Embodiment 81. The method of Embodiment [00403], further comprisingintroducing one or more polymer precursors into said fluidic device.

Embodiment 82. The method of Embodiment [00403] or Embodiment [00404],wherein forming said polymer matrix comprises supplying energy to saidfluidic device to form said polymer matrix.

Embodiment 83. The method of Embodiment [00405], wherein said energy isselectively supplied to one or more portions of said fluidic device.

Embodiment 84. The method of any one of Embodiments [00403] to [00406],further comprising a spatial energy modulating element to selectivelysupply said energy to said fluidic device.

Embodiment 85. The method of Embodiment [00407], wherein said spatialenergy modulating element comprises a physical photomask, a virtualphotomask, a physical electrode distribution pattern, or a virtualelectrode distribution pattern.

Embodiment 86. The method of Embodiment [00407], wherein said spatialenergy modulating element comprises a photolithographic mask or adigital micromirror device (DMD) mask.

Embodiment 87. The method of any one of Embodiments [00405] to [00409],wherein said energy is supplied via a light energy source, a heat energysource, an electrochemical energy source, or an electromagnetic energysource.

Embodiment 88. The method of any one of Embodiments [00405] to [00410],wherein said polymer matrix is coupled to said first surface or saidsecond surface.

Embodiment 89. The method of any one of Embodiments [00405] to [00411],wherein said energy forms said polymer matrix around a portion of saidcoupled biological component.

Embodiment 90. The method of any one of Embodiments [00403] to [00412],wherein at least a portion of said biological component is encapsulatedby said polymer matrix.

Embodiment 91. The method of Embodiment [00413], wherein an entirety ofsaid biological component is encapsulated by said polymer matrix.

Embodiment 92. The method of any one of Embodiments [00403] to [00414],further comprising coupling a first biological component to a firstcapture element to form a first analysis chamber and coupling a secondbiological component to a second capture element to form a secondanalysis chamber.

Embodiment 93. The method of Embodiment [00415], wherein said firstanalysis chamber is adjacent to said second analysis chamber.

Embodiment 94. The method of Embodiment [00415] or Embodiment [00416],wherein said first analysis chamber is disposed from 5 micrometer (μm)to 1,000 μm away from said second analysis chamber.

Embodiment 95. The method of any one of Embodiments [00415] to [00417],further comprising analyzing said first biological component in saidfirst analysis chamber and analyzing said second biological component insaid second analysis chamber.

Embodiment 96. The method of any one of Embodiments [00415] to [00418],further comprising actuating a first reaction in said first biologicalcomponent and actuating a second reaction in said second biologicalcomponent.

Embodiment 97. The method of Embodiment [00419], wherein said firstreaction and said second reaction are different.

Embodiment 98. The method of any one of Embodiments [00415] to [00420],further comprising actuating a third reaction in said first biologicalcomponent and actuating a fourth reaction in said second biologicalcomponent.

Embodiment 99. The method of Embodiment [00421], wherein said thirdreaction and said fourth reaction are different.

Embodiment 100. The method of any one of Embodiments [00403] to [00422],further comprising obtaining a genome, transcriptome, proteome,epigenome, methylome, secretome, or metabolome of said coupledbiological component.

Embodiment 101. The method of Embodiment [00423], wherein the proteomecomprises secreted proteins, surface proteins, or a combination thereof.

Embodiment 102. The method of Embodiment [00423], wherein saidtranscriptome is a substantially full-length transcriptome.

Embodiment 103. The method of Embodiment [00423], wherein saidtranscriptome is a full-length transcriptome.

Embodiment 104. The method of any one of Embodiments [00403] to [00426],further comprising sequencing at least one nucleic acid of saidbiological component.

Embodiment 105. The method of Embodiment [00427], wherein saidsequencing does not comprise amplification of a sequencing library.

Embodiment 106. The method of Embodiment [00428], wherein said nucleicacid library from said biological component is sequenced within a samechamber.

Embodiment 107. The method of any one of Embodiments [00403] to [00428],further comprising coupling a barcode to said biological component or amolecule produced by said biological component.

Embodiment 108. The method of any one of Embodiments [00403] to [00430],further comprising exposing said biological component or said coupledbiological component to an analyte.

Embodiment 109. The method of Embodiment [00431], wherein saidbiological component comprises one or more microbes, and wherein saidanalyte comprises an antimicrobial agent or a microbial growth promotingagent.

Embodiment 110. The method of Embodiment [00432], further comprisingscreening said one or more microbes for susceptibility to saidantimicrobial agent.

Embodiment 111. The method of Embodiment [00431], wherein said analytecomprises a pharmaceutical agent.

Embodiment 112. The method of Embodiment [00434], further comprisingscreening an effect of said pharmaceutical agent on said biologicalcomponent.

Embodiment 113. The method of any one of Embodiments [00403] to [00431],wherein said method further comprises screening said biologicalcomponent for production of a target molecule.

Embodiment 114. The method of Embodiment [00436], wherein said targetmolecule comprises at least one of an antibody, a cytokine, a chemokine,a protein, an antibody derivative, an antibody fragment, a carbohydrate,a toxin, or an aptamer.

Embodiment 115. The method of any one of Embodiments [00403] to [00437],further comprising forming said polymer matrix around said biologicalcomponent such that said biological component is disposed within astructure formed by said polymer matrix.

Embodiment 116. The method of any one of Embodiments [00403] to [00438],further comprising analyzing a local parameter in said first analysischamber or said second analysis chamber, wherein a level of said localparameter in said first analysis chamber is different from a level ofsaid local parameter in said second analysis chamber.

Embodiment 117. The method of Embodiment [00439], wherein said localparameter comprise a pH, an oxygen concentration, or a CO₂concentration.

Embodiment 118. The method of any one of Embodiments [00403] to [00440],wherein said one or more capture elements comprise a polymer matrix.

Embodiment 119. The method of Embodiment [00441], wherein the polymermatrix comprises a hydrogel.

Embodiment 120. A method of obtaining a transcriptome of a biologicalcomponent, said method comprising: (a) forming a polymer matrix on oradjacent to said biological component to form an analysis chamber; and(b) performing one or more reactions in said analysis chamber to obtainsaid transcriptome of said biological component, wherein said biologicalcomponent remains in said analysis chamber during performance of saidone or more reactions.

Embodiment 121. The method of Embodiment [00443], further comprisingcoupling said biological component to a capture element disposed in afluidic device to yield a coupled biological component.

Embodiment 122. The method of Embodiment [00443] or Embodiment [00444],further comprising providing energy from an energy source to saidfluidic device to form said polymer matrix.

Embodiment 123. The method of Embodiment [00445], wherein said energy isprovided selectively using a spatial energy modulating element.

Embodiment 124. The method of Embodiment [00446], wherein said spatialenergy modulating element is generated based on a location of saidbiological component.

Embodiment 125. The method of Embodiment [00446] or Embodiment [00447],wherein said spatial energy modulating element comprises a physicalphotomask, a virtual photomask, a physical electrode distributionpattern, a virtual electrode distribution pattern, a photolithographicmask, or a digital micromirror device (DMD) mask.

Embodiment 126. The method of any one of Embodiments [00443] to [00448],wherein said biological component comprises RNA.

Embodiment 127. The method of any one of Embodiments [00443] to [00449],wherein said RNA is from 50 bases to 100 kb (kilobase bases).

Embodiment 128. The method of any one of Embodiments [00443] to [00450],wherein said polymer matrix comprises pores that are sized to allowdiffusion of a reagent through said polymer matrix but are too small toallow said RNA to traverse said pores.

Embodiment 129. The method of any one of Embodiments [00443] to [00451],wherein said one or more reactions comprise RNA sequencing.

Embodiment 130. A method of analyzing two or more biological components,said method comprising: (a) introducing a first biological component anda second biological component into a fluidic device; (b) forming apolymer matrix on or adjacent to said first biological component to forma first analysis chamber and forming a polymer matrix on or adjacent tosaid second biological component to form a second analysis chamber,wherein said first analysis chamber is adjacent to said second analysischamber in said fluidic device; and (c) analyzing one or more featuresof said first biological component and said second biological component.

Embodiment 131. The method of Embodiment [00453], wherein said one ormore features comprise a first feature and a second feature, and wherein(c) comprises analyzing said first feature and said second feature ofsaid first biological component in said first analysis chamber.

Embodiment 132. The method of Embodiment [00453] or Embodiment [00454],wherein said first biological component remains in said first analysischamber between analysis of each of said first feature and said secondfeature.

Embodiment 133. The method of any one of Embodiments [00453] to [00455],wherein said one or more features comprises a response to an analyte, aresponse to a pharmaceutical agent, a response to an antimicrobialagent, production of a target compound, production of a target molecule,production of a nucleic acid, or production of a protein.

Embodiment 134. The method of any one of Embodiments [00453] to [00456],wherein said first biological component is in biological communicationwith said second biological component.

Embodiment 135. The method of Embodiment [00457], wherein saidbiological communication generates a biological response in said firstbiological component or in said second biological component.

Embodiment 136. The method of Embodiment [00457], wherein saidbiological communication comprises a molecule comprising a protein, anucleic acid, a cytokine, a chemokine, or a combination thereof,generated by said first biological component or by said secondbiological component.

Embodiment 137. A method for identifying a nucleic acid molecule,comprising: providing a polymer matrix comprising said nucleic acidmolecule, and detecting said nucleic acid molecule in absence of nucleicacid amplification.

Embodiment 138. The method of Embodiment [00460], wherein said nucleicacid molecule is a deoxyribonucleic acid (DNA) molecule.

Embodiment 139. The method of Embodiment [00460] or Embodiment [00461],wherein said polymer matrix forms a chamber localizing said nucleicacid.

Embodiment 140. The method any of any one of Embodiments [00460] to[00462], wherein said chamber is formed on demand.

Embodiment 141. The method of any one of Embodiments [00460] to [00463],wherein said polymer matrix is degraded on demand.

Embodiment 142. The method of any one of Embodiments [00461] to [00464],wherein said DNA is 100 base pairs or greater.

Embodiment 143. The method of Embodiment [00460], wherein said nucleicacid is a ribonucleic acid molecule (RNA).

Embodiment 144. The method of Embodiment [00466], wherein said RNA is 50nucleotides or greater.

Embodiment 145. The method of any one of Embodiments [00460] to [00467],further comprising generating a nucleic acid library from saidbiological component within said chamber.

Embodiment 146. The method of Embodiment [00468], wherein said nucleicacid library is sequenced within said chamber.

Embodiment 147. A method for processing a biological component,comprising determining a genome sequence, a transcriptome, a proteome,or an epigenome in absence of nucleic acid amplification, wherein saidprocessing is performed in a single microfluidic device.

Embodiment 148. The method of Embodiment [00470], wherein said cell isat least partially within a polymer matrix.

Embodiment 149. The method of Embodiment [00470] or Embodiment [00471],wherein said polymer matrix is degraded on demand.

Embodiment 150. The method of any one of Embodiments [00470] to [00472],further comprising determining methylation in said cell.

Embodiment 151. A method comprising identifying a plurality of nucleicacid molecules of a plurality of cells without barcoding individualnucleic acid molecules of said plurality of nucleic acid molecules,wherein extracting said plurality of nucleic acid molecules andidentifying are performed in a single microfluidic device.

Embodiment 152. The method of Embodiment [00474], wherein saididentifying comprises sequencing.

Embodiment 153. The method of Embodiment [00474] or [00475], furthercomprising forming a polymer matrix on or adjacent to individual cellsof said plurality of cells such that said individual cells are separatedfrom one another.

Embodiment 154. The method of Embodiment [00476], further comprisingextracting said individual nucleic acid molecules said individual cells.

Embodiment 155. The method of Embodiment [00477], wherein saidsequencing comprises sequencing said individual nucleic acid moleculeswithin said polymer matrix.

Embodiment 156. The method of Embodiment [00475], wherein saidsequencing comprises next-generation sequencing, short-read sequencing,nanopore sequencing, sequencing by synthesis, sequencing by in-situhybridization, or an optical readout.

Embodiment 157. A method comprising: (a) providing a plurality ofnucleic acid molecules within a plurality of matrices, whereinindividual nucleic acid molecules of said plurality of nucleic acidmolecules are from different cells; and (b) sequencing said plurality ofnucleic acid molecules while said plurality of nucleic acid molecules iswithin said plurality of matrices.

Embodiment 158. The method of Embodiment [00480], wherein said pluralityof matrices is disposed in a fluidic device.

Embodiment 159. The method of Embodiment [00480] or Embodiment [00481],wherein said plurality of matrices comprises a plurality of cells, andwherein said plurality of cells comprises said plurality of nucleic acidmolecules.

Embodiment 160. The method of Embodiment [00480], wherein saidsequencing comprises next-generation sequencing, short-read sequencing,nanopore sequencing, sequencing by synthesis, sequencing by in-situhybridization, or an optical readout.

Embodiment 161. A method of analyzing a biological component, saidmethod comprising: (a) introducing one or more biological componentsinto a fluidic device; (b) disposing a first portion of said one or morebiological components adjacent a first surface or a second surface ofsaid fluidic device; and (c) forming one or more polymer matricesadjacent a first portion of said first surface to localize at least oneof said one or more biological component to said first portion.

Embodiment 162. The method of Embodiment [00484], further comprising:(a) agitating said one or more biological components within said fluidicdevice; (b) disposing a second portion of said one or more biologicalcomponents adjacent said first surface of said fluidic device; and (c)forming one or more polymer matrices adjacent a second portion of saidfirst surface to immobilize at least one of said one or more biologicalcomponent of said second portion.

Embodiment 163. The method of Embodiment [00484] or Embodiment [00485],further comprising identifying a position of at least one of said one ormore biological components such that at least one energy source suppliesenergy to said fluidic device to form said one or more polymer matriceson or adjacent said identified position.

Embodiment 164. A system comprising: a fluidic device comprising: a flowchannel; an analysis channel disposed adjacent to said flow channel,wherein at least one flow inhibition element is disposed within saidflow channel to inhibit flow of a biological component; and a layerdisposed between said flow channel and said analysis channel, whereinsaid layer comprises at least one sealable aperture disposed adjacentsaid at least one flow inhibition element, and wherein said at least onesealable aperture is configured to allow passage of said biologicalcomponent; and at least one energy source in communication with saidfluidic device, wherein said at least one energy source is configured toform a polymer matrix within said analysis channel.

Embodiment 165. The system of Embodiment [00487], wherein a portion ofsaid flow channel is substantially parallel with a portion of saidanalysis channel.

Embodiment 166. The system of Embodiment [00487] or Embodiment [00488],wherein said at least one sealable aperture is configured to transitionfrom a sealed state to an open state.

Embodiment 167. The system of Embodiment [00489], wherein passage ofsaid biological component through said at least one sealable aperture isinhibited in said sealed state, and wherein passage of said biologicalcomponent through said at least one sealable aperture is inhibited insaid sealed state.

Embodiment 168. The system of Embodiment [00489] or Embodiment [00490],wherein, when said at least one sealable aperture is in said sealedstate, said at least one sealable aperture is sealed with at least oneof an agarose gel, a temperature-soluble polymer, anN-isopropylacrylamide (NIPAAm) polymer, a wax compound, or an alginate.

Embodiment 169. The system of any one of Embodiments [00487] to [00491],wherein said flow channel comprises a surface disposed opposite of aflow channel surface of said layer, and wherein at least one of said atleast one inhibition elements extends from said surface toward said flowchannel surface such that flow of said biological component in said flowchannel is inhibited by said at least one inhibition element.

Embodiment 170. The system of any one of Embodiments [00487] to [00492],wherein said analysis channel comprises a surface disposed opposite ofsaid analysis channel surface of said layer.

Embodiment 171. The system of any one of Embodiments [00487] to [00493],wherein said flow channel is removably couplable to said analysischannel.

Embodiment 172. The system of any one of Embodiments [00487] to [00494],wherein said surface of said analysis channel comprises one or morebarcodes.

Embodiment 173. The system of Embodiment [00495], wherein the said oneor more barcodes comprise an oligonucleotide.

Embodiment 174. The system of any one of Embodiments [00492] to 173,wherein said polymer matrix is coupled to at least one of said surfaceof said analysis channel or said analysis channel surface of said layer.

Embodiment 175. The system of any one of Embodiments [00492] to [00497],wherein said polymer matrix extends from said surface of said analysischannel to said analysis channel surface of said layer such that saidpolymer matrix surrounds at least a portion of said biologicalcomponent.

Embodiment 176. The system of any one of Embodiments [00487] to [00498],wherein said at least one energy source is in at least one of opticalcommunication, electrochemical communication, electromagnetic,communication, thermal communication, or microwave communication withsaid fluidic device.

Embodiment 177. The system of any one of Embodiments [00487] to [00499],wherein said at least one energy source, comprises a light generatingdevice, a heat generating device, an electrochemical generating device,an electrode, or a microwave device.

Embodiment 178. The system of any one of Embodiments [00487] to [00500],wherein said biological component comprises a plurality of biologicalcomponents.

Embodiment 179. The system of any one of Embodiments [00487] to [00501],wherein said fluidic device is a microfluidic device or a nanofluidicdevice.

Embodiment 180. The system of any one of Embodiments [00487] to 179,wherein said fluidic device comprises a sequencing flow cell.

Embodiment 181. The system of any one of Embodiments [00487] to 180,wherein said fluidic device is used for nucleic acid sequencing.

Embodiment 182. The system of any one of Embodiments [00487] to [00504],wherein said biological component comprises a cell, a nucleic acid, amicrobiome, a protein, a combination of cells, a spatially-linkedbiological component, or a metabolite.

Embodiment 183. The system of Embodiment [00505], wherein said cell isan animal cell, a plant cell, a fungal cell, a bacterial cell, a tumorspheroid, or a combination thereof.

Embodiment 184. The system of Embodiment [00506], wherein said animalcell is a human cell.

Embodiment 185. The system of Embodiment [00505], wherein said nucleicacid is DNA of 100 base pairs or greater or RNA of 50 bases or greater.

Embodiment 186. The system of any one of Embodiments [00487] to [00508],wherein said polymer matrix comprises a hydrogel.

Embodiment 187. The system of any one of Embodiments [00487] to [00509],wherein said fluidic device further comprises one or more polymerprecursors.

Embodiment 188. The system of any one of Embodiments [00510], whereinsaid one or more polymer precursors comprise hydrogel precursors.

Embodiment 189. The system of any one of Embodiments [00487] to [00511],wherein said polymer matrix comprises a polymer matrix wall having awidth of at least 1 μm.

Embodiment 190. The system of any one of Embodiments [00487] to [00512],wherein said polymer matrix inhibits passage of said biologicalcomponent.

Embodiment 191. The system of Embodiment [00512] or Embodiment [00513],wherein said polymer matrix wall is a hydrogel wall.

Embodiment 192. The system of any one of Embodiments [00487] to [00514],wherein said polymer matrix is degradable.

Embodiment 193. The system of Embodiment [00515], wherein degradation ofsaid polymer matrix is “on demand.”

Embodiment 194. The system of Embodiment [00515] or Embodiment [00516],wherein said polymer matrix is degradable by at least one of: (i)contacting said polymer matrix with a cleaving reagent; (ii) heatingsaid polymer matrix to at least 90° C.; or (iii) exposing said polymermatrix to a wavelength of light that cleaves a photo-cleavablecrosslinker that crosslinks said polymer of said polymer matrix.

Embodiment 195. The system of any one of Embodiments [00489] toEmbodiment [00517], wherein said sealable aperture is transitioned tosaid open state by at least one of: (i) contacting said sealableaperture with a cleaving reagent; (ii) heating said sealable aperture toat least 90° C.; or (iii) exposing said sealable aperture to awavelength of light that cleaves a photo-cleavable crosslinker thatcrosslinks said polymer of said sealable aperture.

Embodiment 196. The system of Embodiment [00517], wherein said polymermatrix comprises a hydrogel, and wherein said cleaving reagent isconfigured to degrade said polymer matrix.

Embodiment 197. The system of Embodiment [00519], wherein said cleavingreagent comprises a reducing agent, an oxidative agent, an enzyme, a pHbased cleaving reagent, or a combination thereof.

Embodiment 198. The system of Embodiment [00519], wherein said cleavingreagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine(TCEP), tris(3-hydroxy propyl)phosphine (THP), or a combination thereof.

Embodiment 199. The system of any one of Embodiments [00487] to [00521],wherein said polymer matrix allows passage of a reagent.

Embodiment 200. The system of any one of Embodiments [00487] to [00522],wherein said polymer matrix comprises pores, wherein a size of saidpores are controlled by changing a composition of said one or morepolymer precursors, said at least one energy source, or a combinationthereof.

Embodiment 201. The system of Embodiment [00522], wherein said reagentcomprises at least one of enzymes, chemicals, oligonucleotides, orprimers having a size of less than 50 base pairs.

Embodiment 202. The system of any one of Embodiments [00522] to [00524],wherein said reagent comprises lysozyme, proteinase K, random hexamers,polymerase, transposase, ligase, catalyzing enzyme, deoxynucleotidetriphosphates, buffers, cell culture media, or divalent cations.

Embodiment 203. The system of any one of Embodiments [00487] to [00525],wherein said polymer matrix comprises pores that are sized to allowdiffusion of a reagent through said matrix but are too small to allowDNA or RNA to traverse said pores.

Embodiment 204. The system of any one of Embodiments [00487] to [00526],wherein said polymer matrix comprises a hydrogel, and wherein saidhydrogel comprises polyethylene glycol (PEG)-thiol, PEG-acrylate,acrylamide, N,N′-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO),polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methylmethacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lacticacid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone(PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid),poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin,alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan,gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate,diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallylether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate,polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate,ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritoltetraacrylate, or combinations or mixtures thereof.

Embodiment 205. The system of Embodiment [00527], wherein said hydrogelcomprises an enzymatically degradable hydrogel, PEG-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/PPO.

Embodiment 206. The system of any one of Embodiments [00492] to [00528],wherein said surface comprises one or more barcodes.

Embodiment 207. The system of Embodiment [00529], wherein said one ormore barcodes comprise an oligonucleotide.

Embodiment 208. The system of any one of Embodiments [00492] to 207,wherein said surface of said analysis channel comprises one or morecompounds configured to bind said biological component.

Embodiment 209. The system of any one of Embodiments [00492] to [00531],wherein said surface of said analysis channel is functionalized with asurface polymer.

Embodiment 210. The system of Embodiment [00532], wherein said surfacepolymer is functionalized with an oligonucleotide.

Embodiment 211. The system of Embodiment [00532], wherein said surfacepolymer is functionalized with an antibody, a cytokine, a chemokine, aprotein, an antibody derivative, an antibody fragment, a carbohydrate, atoxin, an aptamer, or any combination thereof.

Embodiment 212. The system of any one of Embodiments [00492] to [00532],wherein a surface of said polymer matrix is functionalized with anoligonucleotide, an antibody, a cytokine, a chemokine, a protein, anantibody derivative, an antibody fragment, a carbohydrate, a toxin, anaptamer, or any combination thereof.

Embodiment 213. The system of Embodiment [00532], wherein said surfacepolymer comprises polyethylene glycol (PEG), a silane polymer,pyridinecarboxaldehyde (PCA), an acrylamide, an agarose, or acombination thereof.

Embodiment 214. The system of any one of Embodiments [00529] to [00536],further comprising a detector for identifying said one or more of saidbiological components, said one or more barcodes, or a combinationthereof.

Embodiment 215. The system of Embodiment [00537], wherein said detectorcomprises a camera.

Embodiment 216. The system of any one of Embodiments [00487] to [00538],further comprising a stage that holds said fluidic device.

Embodiment 217. The system of any one of Embodiments [00487] to [00539],further comprising a sequencing device for obtaining sequencing data.

Embodiment 218. The system of any one of Embodiments [00487] to [00540],further comprising a spatial energy modulating element to selectivelysupply said energy to said fluidic device.

Embodiment 219. The system of Embodiment [00541], wherein said spatialenergy modulating element comprises a physical photomask, a virtualphotomask, a physical electrode distribution pattern, a virtualelectrode distribution pattern.

Embodiment 220. The system of Embodiment [00541], wherein said spatialenergy modulating element comprises a photolithographic mask or adigital micromirror device (DMD) mask.

Embodiment 221. A method of analyzing a biological component, saidmethod comprising: (a) introducing said biological component into a flowchannel of a fluidic device; (b) inhibiting flow of said biologicalcomponent adjacent to an inhibition element, wherein a sealable apertureis disposed adjacent to said inhibition element; (c) disposing saidbiological component from said flow channel to an analysis channel ofsaid fluidic device; and (d) forming a polymer matrix on or adjacent tosaid biological component in said analysis channel.

Embodiment 222. The method of Embodiment [00544], wherein prior to saiddisposing in (c), said sealable aperture is degraded using at least oneof: (i) contacting said sealable aperture with a cleaving reagent; (ii)heating said sealable aperture to at least 90° C.; or (iii) exposingsaid sealable aperture to a wavelength of light that cleaves aphoto-cleavable crosslinker that crosslinks said polymer of saidsealable aperture.

Embodiment 223. The method of Embodiment [00544] or Embodiment [00545],further comprising introducing one or more polymer precursors into saidfluidic device.

Embodiment 224. The method of any one of Embodiments [00544] to [00546],wherein forming said polymer matrix comprises supplying energy to saidfluidic device to form said polymer matrix.

Embodiment 225. The method of Embodiment [00547], wherein said energy isselectively supplied to one or more portions of said fluidic device.

Embodiment 226. The method of any one of Embodiments [00544] to [00548],further comprising activating a spatial energy modulating element toselectively supply said energy to said fluidic device.

Embodiment 227. The method of Embodiment [00549], wherein said spatialenergy modulating element comprises a physical photomask, a virtualphotomask, a physical electrode distribution pattern, or a virtualelectrode distribution pattern.

Embodiment 228. The method of Embodiment [00549], wherein said spatialenergy modulating element comprises a photolithographic mask or adigital micromirror device (DMD) mask.

Embodiment 229. The method of any one of Embodiments [00547] to [00551],wherein said energy is supplied via light energy source, a heat energysource, an electrochemical energy source, or an electromagnetic energysource.

Embodiment 230. The method of any one of Embodiments [00547] to [00552],wherein said polymer matrix is coupled to a surface of said analysischannel.

Embodiment 231. The method of any one of Embodiments [00547] to [00552],wherein said energy forms said polymer matrix around a portion of saidcoupled biological component.

Embodiment 232. The method of any one of Embodiments [00544] to [00554],wherein at least a portion of said biological component is encapsulatedby said polymer matrix.

Embodiment 233. The method of Embodiment [00555], wherein an entirety ofsaid biological component is encapsulated by said polymer matrix.

Embodiment 234. The method of any one of Embodiments [00544] to [00556],further comprising encapsulating a first biological component to form afirst analysis chamber and encapsulating a second biological componentto form a second analysis chamber.

Embodiment 235. The method of Embodiment [00557], wherein said firstanalysis chamber is adjacent to said second analysis chamber.

Embodiment 236. The method of Embodiment [00557] or Embodiment [00558],wherein said first analysis chamber is disposed from 5 micrometer (μm)to 1,000 μm away from said second analysis chamber.

Embodiment 237. The method of any one of Embodiments [00557] to [00559],further comprising analyzing said first biological component in saidfirst analysis chamber and analyzing said second biological component insaid second analysis chamber.

Embodiment 238. The method of any one of Embodiments [00557] to [00560],further comprising actuating a first reaction in said first biologicalcomponent and actuating a second reaction in said second biologicalcomponent.

Embodiment 239. The method of Embodiment [00561], wherein said firstreaction and said second reaction are different.

Embodiment 240. The method of any one of Embodiments [00557] to [00562],further comprising actuating a third reaction in said first biologicalcomponent and actuating a fourth reaction in said second biologicalcomponent.

Embodiment 241. The method of Embodiment [00563], wherein said thirdreaction and said fourth reaction are different.

Embodiment 242. The method of any one of Embodiments [00544] to [00564],further comprising obtaining a genome, transcriptome, proteome,epigenome, methylome, secretome, or metabolome of said biologicalcomponent.

Embodiment 243. The method of Embodiment [00565], wherein saidtranscriptome is a substantially full-length transcriptome.

Embodiment 244. The method of Embodiment [00565], wherein saidtranscriptome is a full-length transcriptome.

Embodiment 245. The method of any one of Embodiments [00544] to [00567],further comprising sequencing said biological component.

Embodiment 246. The method of Embodiment [00568], wherein saidsequencing does not comprise amplification of a sequencing library.

Embodiment 247. The method of any one of Embodiments [00544] to [00569],further comprising a barcode configured to be coupled to said biologicalcomponent or a molecule produced by said biological component.

Embodiment 248. The method of any one of Embodiments [00544] to [00570],further comprising exposing said biological component or said firstbiological component to an analyte.

Embodiment 249. The method of Embodiment [00571], wherein saidbiological component comprises one or more microbes, and wherein saidanalyte comprises an antimicrobial agent, a microbial growth promotingchemical, or a combination thereof.

Embodiment 250. The method of Embodiment [00572], further comprisingscreening said one or more microbes for susceptibility to saidantimicrobial agent.

Embodiment 251. The method of Embodiment [00571], wherein said analytecomprises a pharmaceutical agent.

Embodiment 252. The method of Embodiment [00574], further comprisingscreening an effect of said pharmaceutical agent on said biologicalcomponent.

Embodiment 253. The method of any one of Embodiments [00544] to [00571],wherein said method further comprises screening said biologicalcomponent for production of a target molecule.

Embodiment 254. The method of Embodiment [00576], wherein said targetmolecule comprises at least one of an antibody, a cytokine, a chemokine,a protein, an antibody derivative, an antibody fragment, a carbohydrate,a toxin, or an aptamer.

Embodiment 255. The method of any one of Embodiments [00544] to [00577],further comprising forming said polymer matrix around said biologicalcomponent such that said biological component is disposed within astructure formed by said polymer matrix.

Embodiment 256. The method of any one of Embodiments [00544] to [00578],further comprising analyzing a local parameter in said first analysischamber or said second analysis chamber, wherein a level of said localparameter in said first analysis chamber is different from a level ofsaid local parameter in said second analysis chamber.

Embodiment 257. The method of Embodiment [00579], wherein said localparameter comprise a pH, an oxygen concentration, or a CO₂concentration.

EXAMPLES

The following illustrative examples are representative of embodiments ofthe devices and methods described herein and are not meant to belimiting in any way.

Example 1: mRNA 3′ Gene Expression Workflow with External Sequencing

This example demonstrates an mRNA 3′ gene expression workflow using afluidic device comprising a hydrogel as described herein. FIGS. 13A-13Cillustrate various steps of the workflow. FIG. 13A shows the process ofloading cells and processing cells to extract mRNAs. Step 1300 showscells that are provided into the fluidic device. A cell 1302 may betrapped using a capture element or a trap 1303 as described herein, suchthat the cell 1302 is contained or immobilized at a location on asurface 1311 of a channel 1304 of the fluidic device (1310). Thetrapping can be performed on the surface 1312. Polymer matrix 1321, 1322is generated adjacent to the cell to form a compartment to localize thecell and parts and/or products thereof (1320). Reagents flow into thechannel and into the polymer matrix compartment, where the polymermatrix is porous and allows transfer of reagents. The reagents areprovided to lyse the cell in order to extract genetic material from thecell (step 1330).

FIG. 13B shows an mRNA molecule 1339 that is extracted from the cellwithin the polymer matrix, as described herein. The mRNA 1339 iscaptured using a capture element that comprises various portions. Inthis non-limiting example, the capture element shown in step 1340comprises an oligo 1341, a unique molecular identifier (UMI) 1342, abarcode (BC) 1343, and sequencing primers 1344, 1345. The oligo 1341 cancomprise a plurality of thymine bases (e.g., 30 thymine bases) to bindto the poly-A tail of the mRNA. The UMI 1342 is used to maintain theuniqueness of the captured mRNA during the downstream amplificationsteps. The BC 1343 is used to associate the mRNA to the cell that it wasextracted from. Furthermore, the BC could be used to associate furtherinformation about the source of the mRNA sequence such as the samplethat the cell was obtained from. The sequencing primer 1344, 1345 maycomprise an R1 and/or a P7 primer.

After capturing the mRNA molecule (step 1340), reverse transcription isperformed to copy the mRNA into cDNA then integrate the UMI 1342, the BC1343, and the sequencing primers 1344, 1345 into the cDNA (complementaryDNA) molecule (step 1350) to generate a barcoded cDNA strand thatcarries information regarding, for example, the cell and/or the sampleit was extracted from. In step 1360, template switch oligos (TSO) 1361hybridize to untemplated C nucleotides 1351 added during reversetranscription in step 1350. The cDNA is then extended and denatured toform a single-strand DNA as shown in step 1370. Then the library isamplified (e.g., via PCR) (step 1375). The library produced in steps1300 to 1375 is then eluted (step 1380) and transferred out of thefluidic device and pooled together as shown in 1390. The eluted cDNA isprocessed by fragmentation and adapter ligation (e.g., throughtagmentation) to add sequencing adapters including specific sequencesdesigned to interact with the sequencing platform used for sequencing aswell as sample indexes (step 1395). The polymer matrix may bedeconstructed after steps 1340, 1350, 1360, 1370, or 1375.

Example 2: mRNA 3′ Gene Expression Workflow with Internal Sequencing

The mRNA gene expression assay can be performed using the fluidic devicethat is used for extracting and capturing the mRNA from a cell localizedby using a polymer matrix formed as described herein. FIGS. 14A and 14Billustrate an example for the steps of preparing a library forsequencing to be performed in the fluidic device. A cell is providedinto the fluidic channel, cell is localized by trapping and/or formingpolymer matrix, and the mRNA is extracted by cell lysis similar to steps1300, 1310, 1320, 1330 provided in FIG. 13A in Example 1. The mRNA 1401extracted from the cell are then captured using a capture element 1402.In this non-limiting example, the capture element 1402 shown in step1400 comprises an oligo 1403, and sequencing primers 1404, 1405 (e.g.,R1 or P7 primers). The oligo 1403 may comprise a plurality of thyminebases 1403 (e.g., 30 thymine bases) to bind to the poly-A tail of themRNA 1401. As shown in this example, a UMI or a barcode is not requiredas the sequencing is performed on the fluidic device as opposed to anexternal sequencing device. The spatial information (e.g., location ofthe polymer matrix and capturing site) associates the mRNA with the cellthat generated the mRNA. In this process, the sequencing readouts sharethe same location and therefore are associated with the cell, which themRNA was extracted from. After capturing the mRNA molecule (step 1400),reverse transcription is performed to copy the mRNA into cDNA (step1410). In step 1420, template switch oligos (TSO) 1421 hybridize tountemplated C nucleotides 1415 added during reverse transcription instep 1410. The cDNA is then extended to form a double-strand DNA asshown in step 1430.

FIG. 14B illustrates the remainder of the process steps. This includes atagmentation step or fragmentation and adapter ligation step as shown instep 1440. Then the end tail of the cDNA is hybridized onto theappropriate surface primer (step 1450). The hybridization is followed byclustering (surface amplification) the cDNA molecules (step 1460). Theclustered cDNA molecules are linearized to generate single strandedmolecules (step 1470). Sequencing is then performed in situ inside thefluidic device. This process does not require an amplification step, asthe linearized cDNA molecules are not eluted off the surface of thefluidic device to be sent to another device for sequencing thus reducingDNA molecule losses. The association of the cDNA molecules with thelocation of the cell on the surface of the fluidic device may eliminatethe use of any barcoding or UMI for tracking the source of thesequencing products/read outs. In this example, the sequencing can beshort-read sequencing, nanopore sequencing, sequencing by synthesis,sequencing by hybridization, or sequencing through collection of anyoptical readouts.

Example 3: Whole Genome Workflow

FIG. 18 schematically illustrates a whole genome workflow followed byexternal sequencing. A biological component (e.g., a cell) is providedinto a fluidic channel as described here, the whole genome material(e.g., DNA) is extracted by capturing and lysing a biological componentusing polymer matrix compartments similar to steps: 1300, 1310, 1320,and 1330 provided in FIG. 13A as discussed in Example 1. DNA may beextracted from a biological as shown in step 1801 of FIG. 18. In step1801 a double-stranded DNA library may be generated. Step 1801 maycomprise tagmentation, and/or enzymatic fragmentation and adapterligation. In step 1801, DNA extracted from the biological component maybe fragmented into fragments of predefined sizes 1811. Step 1801 mayfurther comprise inserting one or more adapters 1803/1804 (e.g., asequencing primer R2) to one or both ends of the DNA fragments. Adenatured DNA fragment 1811 carrying one or more adapter sequences maythen be captured on a surface 1802 of the fluidic channel using acapture element (step 1810). The capture element may comprise barcodingregion 1813, sequencing primers 1814, 1815 (e.g., R1/R2 or P5/P7primers), and capturing sequences 1812. The captured DNA may then betagged, extended (step 1820) and amplified (e.g., via PCR) (step 1830).The barcode region 1813 of the capture element may ensure that thegenomic material of a biological component may be uniquely tagged andlater identified using, for example, sequencing. An amplified DNAprepared in step 1830 may then be eluted from the surface andtransferred out of the fluidic device in step 1840. Additional primersor sample indexes may be added to the eluted molecules to prepare asequencing library (step 1850). The eluted genomic material may then besequenced using a device different than or separate from the fluidicdevice described herein. In some cases, the genomic material may besequenced in and/or using the fluidic device described herein, wheresteps of elution of the genomic material and barcoding may beeliminated. The hydrogel can be dissolved after any of the steps 1810,1820, or 1830.

Example 4: Multi-Omics Workflow

FIG. 19 illustrates an example of a workflow for multi-omic analysis ofa sample comprising a biological component. In some cases, thebiological component may comprise a cell. The cell may be an animal cell(e.g., a human cell), a plant cell, a fungal cell, a bacterial cell, orany other type of cell capable of producing proteins. In step 1901,biological components can be collected to be introduced into or providedto a fluidic device. In some embodiments, a capture step 1902 may usecapture elements, as described herein, to capture and/or couple abiological component to a surface of a channel disposed within thefluidic device. Capture elements may comprise a functional group,fibronectin, RGD peptides, antibodies, or other molecules as describedherein. The capture element may be capable of capturing or coupling thebiological component or suspected biological component. In anencapsulation step 1903 a polymer matrix may be formed adjacent to thebiological component. In some embodiments, the biological component maybe encapsulated entirely by the polymer matrix (e.g., hydrogel) or aportion thereof. In another embodiment, the biological component isencapsulated by the pattern of the hydrogel, or a portion thereof, andone or more surfaces of the fluidic device or channel. The polymermatrix (e.g., hydrogel) may be porous and allows transfer of reagents.

A reagent may comprise a cell incubation solution. The reagent may beprovided into the channel and into the polymer matrix compartment. Thecell incubation reagent may promote the biological component to growthand/or to produce and secrete a secretome. In some embodiments, asecretome comprising antibodies produced and secreted from thebiological component may be localized within the polymer matrix (step1904). In step 1905 proteins produced by and/or secreted from thebiological component may be captured and tagged. A molecular barcode(e.g., fluorescent labeled antibody) may be used for tagging theprotein. In step 1906, the biological component may be lysed to releasegenetic material (e.g., mRNA) from within the biological component, forexample, by introducing of a lysis buffer into the polymer matrixcompartment. In certain embodiments, an average pore size of the polymermatrix may permit, or may be induced to permit, the flow of reagentsinto the polymer matrix compartment. In step 1907, the genetic materialreleased from the biological component may be captured by capture agentson a surface of the channel. The hydrogel may then be dismantled toallow further chemical processing (step 1908). For example, the capturednucleic acids may comprise ribonucleic acids (e.g., mRNA). Reversetranscription may be performed to generate a complementary DNA (cDNA)and cDNA may be extended in step 1909. In step 1910, template switchingcan be performed to add additional primers to cDNA molecules. Templateswitching may occur prior to further denaturation and extension (step1911). The resulting nucleic acid strands may be tagmented (step 1912).The protein tags and cDNA molecules may be amplified (steps 1913 and1914) and then eluted from the surface of the channel eithersequentially or in parallel (e.g., simultaneously). The genomic andproteomic material may then be pooled (step 1915) and further processed.Further processing may comprise nucleic acid amplification, addition ofmore primers, gene sequencing, and/or protein analysis processes. Insome embodiments, additional primers comprising sequencing primers(e.g., P5/P7 primers) and sample index may be added to the libraries ofnucleic acids generated by the method described herein before sequencingthe libraries.

Example 5: Single Cell Detection Using Discrete Capture Mode

Jurkat cells (immortalized line of human T lymphocyte cells) can firstbe stained with Calcein AM dye for fluorescence imaging. The cells canbe mixed with the hydrogel precursor mix containing hydrogel monomer,cross-linker and photo-initiator. The ratio of single-cell suspensionand hydrogel precursor mix may be titrated until a designed cell density(e.g. ˜30 cells/ul) is obtained. After loading single-cell hydrogelprecursor mix to the microfluidic channel, the cells will settle down tothe bottom surface of the microfluidic channel. The cells can then beimaged using a microscope. The image may be processed to identify thelocation of all the cells in the image. A virtual mask can be generatedsuch that each cell is encapsulated inside a circular ring of approx.180 um outer diameter and 30 um wall width. The virtual mask may then beprojected using a DMD projector and a 10× magnification microscopeobjective. The UV light pattern corresponding to the virtual mask can beprojected for 6 seconds and can result in the hydrogel structure shownin FIG. 25A. After patterning the hydrogel, the excess monomer andphoto-initiator can be washed away with cell-compatible buffer.

Example 6: Single Cell Detection Using Contiguous Capture Mode

Jurkat cells are mixed with the hydrogel precursor mix containinghydrogel monomer, cross-linker and photo-initiator. The ratio ofsingle-cell suspension and hydrogel precursor mix is titrated untildesigned cell density (e.g. ˜30 cells/ul) is obtained. After loadingsingle-cell hydrogel precursor mix to the microfluidic channel, thecells will settle down to the bottom surface of the microfluidicchannel. After the cells are identified using bright field imaging, thecenters of cells are identified, and a virtual mask is generated using aVoronoi algorithm that results in a non-circular shaped hydrogel matrixstructure (e.g., a particular CellCage™ structure) with a single wallseparating two adjacent cells that are closer than a specified distancethreshold. FIG. 25B shows cells identified using bright field imagingusing 4× microscope objective, the resulting Voronoi mask calculatedbased on the location of the cells, and the hydrogel structuressurrounding the cells generated using the Voronoi mask.

Example 7: Single Cell Detection and Retention

FIG. 26A illustrates the selective retention of fluorescent cells in amixture of fluorescent calcein AM stained cells and non-fluorescentcells. The mixture of mammalian cells can include Calcein AM stained andunstained cells and may be agitated gently in cell-compatible buffer toavoid cell aggregation. The single-cell suspension may be added into ahydrogel precursor mix that contains monomer, cleavable cross-linker andphoto-initiator with cell-compatible buffer condition. The ratio ofsingle-cell suspension and hydrogel precursor mix is titrated untilgetting designed cell density (e.g. ˜30 cells/ul). Upon loadingsingle-cell hydrogel precursor mix to the microfluidic channel, thecells settle down to the bottom surface. The cells can be imaged usingbrightfield and fluorescence mode, and their location is identifiedusing image processing. A virtual mask may be generated to encapsulateonly the fluorescent cells. The UV light with the specific pattern canbe applied to the channel to cross-link the hydrogel within the lightpath. As shown in FIG. 26A, the ring-shaped hydrogel can be formedaround fluorescent cells. After patterning the hydrogel, the excessmonomer, photo-initiator and non-captured cells (non-fluorescent) can bewashed away with cell-compatible buffer. Only fluorescent single cellsare retained, and the cells will be ready for various assays and cellmRNA/DNA/protein content analysis.

FIG. 26B illustrates selective retention of cells of interest andremoval of cells that are not desired. Following the performance ofvarious cell assays, the cells of interest, after a first round ofencapsulation, can be retained by making a concentric ring hydrogelstructure using a non-cleavable gel around the previously formedcleavable hydrogel matrix. The non-cleavable hydrogel precursor mix thatcontains monomer, non-cleavable cross-linker and photo-initiator may beloaded into the microfluidic channel containing hydrogel matrices. Thespecific pattern of UV light may be applied to form non-cleavablehydrogel rings on the selected hydrogel matrices. After washing away thenon-cleavable hydrogel precursor mix, hydrogel cleaving reagents may beloaded to the microfluidic channel. This will result in the melting ofthe hydrogel matrix. The cells that did not get selected will not becompartmentalized and will instead be washed away. This will result inthe cell retention for the cells of interest.

Example 8: Single-Cell Transcriptomic Workflow (Jurkat Cells)

Jurkat cells (immortalized line of human T lymphocyte cells) can beagitated gently in the cell-compatible buffer to avoid aggregation. Thesingle-cell suspension can then be added into a hydrogel precursor mixthat contains monomer, cleavable cross-linker and photo-initiator withcell-compatible buffer condition. The ratio of single-cell suspensionand hydrogel precursor mix can be titrated until designed cell densityis obtained. After loading single-cell hydrogel precursor mix to themicrofluidic channel, the cells settle down to the bottom surface. Aspecific pattern of UV light may be applied to the channel to cross-linkthe hydrogel within the light path. As shown in FIG. 27A, a ring-shapedhydrogel matrix can be formed around each single cell. After patterningthe hydrogel, the excess monomer and photo-initiator can then be washedaway with cell-compatible buffer.

The top and/or bottom surface of a flow cell can be coated with an Oligolawn consisting of poly-T mRNA capture oligos and surface amplificationprimers. A cell lysis buffer consisting of 200 mM Tris pH7.5, 20 mMEDTA, 2% sarcoyl, 6% Ficoll can be introduced into the channel and afterlysis, the mRNA molecules released from the cell can be captured by thepoly-T capture oligos on both top and bottom surface with mRNA poly-Atails hybridization. The hydrogel of the hydrogel matrix is designed tohave pore size that prevents leaking of the mRNA outside the hydrogelmatrix, so all of the mRNA molecules stay inside the hydrogel matrixuntil they are fully captured by the poly-T capture oligos. A hydrogelcleaving reagent is then loaded into the channel to dissolve thehydrogel without disturbing all mRNA molecules hybridized to thesurface. The captured mRNA is converted to DNA library. A reversetranscription reagent (consisting of Maxima H+ reverse transcriptase in1×RT buffer), template switch oligo, SUPERase-In RNase Inhibitor, anddNTPs can be loaded into the flow cell to synthesize the complementarycDNA from the mRNA molecules captured by the poly-T capture oligoanchored on the surface. The 2nd-strand cDNA is then synthesized,followed by a tagmentation reaction with a Tn5 transposase enzyme tofragment the cDNA & introduce the amplification primer to the 5′-end ofthe 2nd Stranded cDNA molecule. Only the anchored cDNA fragmentscorresponding to the 1st strand synthesized cDNA will be retained. Atthis step, all of the captured mRNA molecules on the surface will beconverted to the cDNA library. After this, bridge amplification isperformed using the surface amplification primers present on the surfacein proximity to cDNA library molecules. The resulting clusters of eachcDNA molecule on the surface are then sequenced using a sequencing bysynthesis method to decode the sequence of single cell mRNA moleculeswithin a compartment.

FIG. 27A shows hydrogel matrices inside a fluidic channel with a singlecell inside each hydrogel matrix. FIG. 27B is a spatial plot showing thelocation of DNA clusters with sequences aligning to mRNA molecules fromJurkat cells. Regions with a high density of mapped DNA clusters in theplot align with the location of hydrogel matrices, and mRNA reads withineach such region corresponds to mRNA released from the single cell. Thefastq files from sequencing can be mapped using STAR aligner to hg38genome, and the x,y location of each aligned cluster within a tile isplotted to generate the spatial plot. All the DNA clusters within eachhydrogel matrix is assigned a unique barcode corresponding to theidentity cell that was present in it. The left panels of FIGS. 27C and27D shows two highlighted hydrogel matrices with a cell inside eachusing a higher magnification image. The right panels of FIGS. 27C and27D show the corresponding spatial plot of DNA sequences mapping tohuman mRNA.

Example 9: Single Cell mRNA (Jurkat and Mouse) Sequencing and Analysis

The dataset in FIG. 28A corresponds to single cell mRNA sequencing usinga mixture of human and mouse cells freshly obtained from cell cultures,according to some embodiments. Cells can be washed with 1×PBS-0.04%BSA-1% SUPERase-In. The concentration of cells can be adjusted using1×PBS-0.04% BSA 1% SUPERase-In to have similar cell count to respect toeach cell population and to have a single cell per hydrogel matrix. Anequal mixture of human and mouse cells is added into the hydrogelprecursor mix and encapsulated to achieve one cell per hydrogel matrix.Then, the cell can be lysed using a cell lysis buffer consisting of 200mM Tris pH7.5.20 mM EDTA, 2% sarcoyl, and 6% Ficoll. Subsequently, aftercDNA synthesis and tagmentation of the cDNA molecule, the resulting DNAlibrary can be amplified using bridge amplification, and the DNA librarycan be sequenced.

The fastq files from sequencing data can be mapped to a combined humanand mouse genome and aligned using STAR aligner. The reads mappinguniquely to human or mouse genome can be extracted, and the location ofthe mapped reads within a tile can be plotted to obtain spatial plots ofreads location. High density regions of mapped regions corresponding tohydrogel matrices are assigned a barcode. The number of reads uniquelymapping to mouse and human genome within each hydrogel matrix iscalculated and plotted in the scatterplot shown in FIG. 28A. Each pointin the scatterplot corresponds to a hydrogel matrix barcode and showsthe number of reads mapping uniquely to human or mouse genome. Themajority of reads are primarily from human or mouse genome, indicatingsingle cell capture within each hydrogel matrix. FIG. 28B shows thecorresponding spatial plots of DNA sequences mapping to human and mousemRNA.

Example 10: Single Cell Surface Protein Workflow

Frozen PBMC (peripheral blood mononuclear cells) cells, which can befreshly obtained or previously fixed, can be thawed gently in awaterbath at 37° C. Cells can be diluted further in RPMI medium to washaway dimethyl sulfoxide or any other solvents. Then, the PBMC can bewashed and resuspended in appropriate buffer. The PBMC can first beincubated in blocking solution to reduce nonspecific binding, then thecells can be labelled with a proper antibody panel (each antibody isflanked with an oligo sequence with barcode and polyA tail, as shown inFIG. 29A) in tube. After the labelling step, cells can be washed well toget rid of excess antibodies and filtered to remove aggregations. Thecell concentration is adjusted for cage array, so there is one cell percage. Labeled cells can be added into a hydrogel precursor mix thatcontains monomer, cleavable cross-linker and photo-initiator withcell-compatible buffer condition. The cells can be loaded into theflowcell along with the hydrogel precursor mix and encapsulated toachieve one cell per hydrogel matrix. Cells are lysed with lysis buffer,and labelled cell surface proteins are released in the hydrogel matrix.

The top and bottom surfaces of the hydrogel matrix can have a dense lawnof poly-T mRNA capture oligos that capture the polyA tails ofantibody-cell surface protein complexes. The hydrogel of the hydrogelmatrix can be designed to have a pore size that prevents leaking of theantibody-labelled cell proteins outside the hydrogel matrix, so all ofthe labelled cell surface proteins stay inside the hydrogel matrix untilthey are fully captured by the surfaces. The hydrogel cleaving reagentcan then be loaded to the channel to dissolve the hydrogel withoutdisturbing labelled and captured protein molecules from the top andbottom surfaces. Then captured oligo sequence can be copied on thesurface using an extension mix consisting of DNA polymerase and dNTP.After copying, the original molecules can be washed away with 0.1N NaOHto retain a surface bound single stranded molecule. After bridgeamplification using the surface amplification primers surrounding thecopied molecule, a cluster of each captured oligo can be formed, and thesurface is ready for sequencing to decode the content of cell surfaceprotein.

FIG. 29B shows the relative expression of various surface proteinsdetected after sequencing the antibody barcode oligos captured afterlysing cells inside the hydrogel hydrogel matrix. The cells areclassified into various cell types (CD8+T cells, monocytes, CD4+T cells,NK cells, DC cells, and B cells) based on the relative expression ofthese antibodies on the cell surface.

FIG. 29C shows the representative cells inside cell cages and spatialplots of barcode sequence, color coded by the identity of the barcodesequence, illustrating abundance of various antibodies inside eachhydrogel matrix after cell lysis.

Example 11: CHO Cell Secretion and mRNA Workflow

The hydrogel matrix can be used for single-cell secretion proteindetection since it provides an enclosed and static environment for eachsingle cell. To analyze the IgG molecules produced and secreted fromeach single CHO DP-12 cell, detection beads (3 um-diameter streptavidinbeads carrying the biotinylated Antibody for IgG binding) and cellcapture beads (3 um-diameter streptavidin beads carrying the fibronectinmolecules) can be first immobilized on the surface of the microfluidicdevice with amplification and poly-T mRNA capturing oligos. CHO DP-12cells can then be loaded into the microfluidic device and immobilized bythe fibronectin coated beads. Specific cell culture media for CHO DP-12cells can then then loaded into the microfluidic device and thenincubated at 37° C. for about 2-4 hours. Each CHO DP-12 cell willsecrete IgG molecules, which can be collected by IgG antibodies on the3-um streptavidin beads adjacent to the cell. After incubation, thesecondary detection antibody for IgG with fluorescent dye can be loadedto the microfluidic channel. The 3-um streptavidin beads that havecaptured the IgG molecules secreted from the CHO DP-12 cells will lightup in the fluorescent emission images, as shown in FIG. 30A. Thefluorescent signal can then be used to determine the IgG secretion levelfor each single CHO DP-12 cell.

Following the IgG detection process, the single CHO-cell mRNA can bedirectly sequenced in the same hydrogel matrix. A cell lysis buffer canbe introduced into the channel. After the cell is lysed, the mRNAmolecules released from the cell can be captured by the poly-T captureoligos on both the top and bottom surfaces via mRNA poly-A tailshybridization. The hydrogel of the hydrogel matrix can be designed tohave pore size that prevents the mRNA from leaking outside of thehydrogel matrix, so all the mRNA molecules stay inside the hydrogelmatrix until they are fully captured by the poly-T capture oligos on thetop and bottom surfaces. The hydrogel cleaving reagent can then beloaded into the channel to dissolve the hydrogel without disturbing allthe mRNA molecules hybridized to the surface. The captured mRNA can beconverted to a DNA library. A reverse transcription reagent consistingof Maxima H+ reverse transcriptase in 1× buffer can be loaded into theflow cell to synthesize the complementary cDNA from the mRNA moleculescaptured by the poly-T capture oligo anchored on the surface. The2nd-strand cDNA can then be synthesized. Next, Tn-5 tagmentation canfragment the cDNA and introduce the amplification primer to the 3′-endof the 1st Stranded cDNA molecule. Only the anchored cDNA fragments(close to 3′ end of the original mRNA molecule) will be retained. Atthis step, all of the captured mRNA molecules on the surface will beconverted to the cDNA library. After this, bridge amplification can beperformed using the surface amplification primers present on the surfacein proximity to cDNA library molecules. The resulting clusters of eachcDNA molecule on the surface can then be sequenced using a sequencing bysynthesis method to decode the sequence of single cell mRNA moleculeswithin a compartment.

Example 12: IL-2 Secretion and mRNA Workflow

IL2-secreting cells obtained from cell culture can be counted, and thenthe concentration can be adjusted to 1-2M cells per 1 mL in cell culturemedium. 2 ul of Cell Activation Cocktail from BioLegend (withoutBrefeldin A) per 1 mL can be added to the cell culture, and the cellculture flask is placed in a 37° C. incubator for 3-4 hours to stimulatecells to secrete IL-2. The stimulated cells can then be washed with1×PBS-0.04% BSA and mixed with hydrogel precursor mix to form hydrogelmatrices. After cell encapsulation, cells can be washed and incubatedfurther in a cell culture medium with Cell Activation Cocktail (2 uL ofcocktail in 1 mL of cell culture medium).

An IL-2 bead-based ELISA kit can be obtained from a provider likeBioLegend Inc. The bead, containing IL-2 capture antibodies, can beloaded to the microfluidic channel in PH 6.1 MES buffer to allowimmobilization of the bead via surface static charges. The microfluidicchannel can then be washed with 1×PBS buffer. The APTES in 1×PBSsolution (0.25%) can then be loaded into the microfluidic channel tocreate a positively charged surface for the following cellimmobilization. Stimulated Jurkat E6.1 cells in specific cell culturemedia containing the IL-2 stimulation chemicals for Jurkat E6.1 cellscan then be loaded into the microfluidic device followed by 37° C.incubation for 4 hours. Each Jurkat E6.1 cell inside the hydrogel matrixwill start to secrete IL-2 molecules, which will be collected by theIL-2 capture antibodies on the BioLegend ELISA beads adjacent to thecell. After incubation, the secondary detection antibody for IL-2labeled with biotin can be loaded to the microfluidic channel. The ELISAbeads that have captured the IL-2 molecules secreted form the Jurkatcells will light up in the fluorescent emission images after incubationof streptavidin fluorescent dyes. The fluorescent signal can be used todetermine the IL-2 secretion level for each single cell.

Following the IL-2 detection process, the single Jurkat-cell mRNA can bedirectly sequenced in the same microfluidic channel. Hydrogel precursormix that contains monomer, cleavable cross-linker and photo-initiatorwith cell-compatible buffer condition can be loaded to the microfluidicchannel. A specific pattern of UV light can be applied to the channel tocross-link the hydrogel within the light path. A ring-shaped hydrogelcan be formed around each single cell. The hydrogel functions as ahydrogel matrix. A cell lysis buffer can be introduced into the channel,and after lysis, the mRNA molecules released from the cell can becaptured by the poly-T capture oligos on both top and bottom surfacesvia mRNA poly-A tails hybridization. The hydrogel of the hydrogel matrixcan be designed to have pore size that prevents the mRNA from leakingoutside of the hydrogel matrix, so all of the mRNA molecules remaininside the hydrogel matrix until they are fully captured by thesurfaces. The hydrogel cleaving reagent can then be loaded into thechannel to dissolve the hydrogel without disturbing all mRNA moleculeshybridized to the surface.

The captured mRNA is converted to DNA library. A reverse transcriptionreagent consisting of Maxima H+ reverse transcriptase in 1× buffer canbe loaded into the flowcell to synthesize the complementary cDNA fromthe mRNA molecules captured by the poly-T capture oligo anchored on thesurface. The 2nd-strand cDNA can then be synthesized. Next, Tn-5tagmentation can fragment the cDNA and introduce the amplificationprimer to the 3′-end of the 1st Stranded cDNA molecule. Only theanchored cDNA fragments (close to 3′ end of the original mRNA molecule)will be retained. At this step, all of the captured mRNA molecules onthe surface will be converted to the cDNA library. After this, bridgeamplification can be performed using the surface amplification primerspresent on the surface in proximity to cDNA library molecules. Theresulting clusters of each cDNA molecule on the surface can then besequenced using a sequencing by synthesis method to decode the sequenceof single cell mRNA molecules within a compartment.

FIG. 31B shows the gene expression analysis of non-stimulated &stimulated Jurkat cells. Upon stimulation, several of the key markergenes such as CD69, GZMB, NFKB1A, DUSP2 are upregulated and severalgenes such as BCL11B, NOTCH3, MYO7B are downregulated. A fraction of thestimulated cells secrete IL2. FIG. 31C shows the hydrogel matricescorresponding to IL-2 secreting cells using the secreted proteinquantification based on ELISA as well as hydrogel matrices with mRNAreads mapping to IL2.

Example 13: CHO Cell Culture in Dextran Chamber

A hydrogel matrix can be used for cell culture if a cell-compatiblehydrogel wall material is applied and there is a proper bottom surfacefor cell attachment. To culture CHO cells in a hydrogel matrix, apoly-L-lysine substrate can be used for microfluidic channelconstruction (poly-L-lysine is a positive charged layer widely used forCHO cell culture). The CHO cells can be loaded to the microfluidicchannel in hydrogel precursor mix that contains monomer (dextran),cleavable cross-linker and photo-initiator with cell-compatible buffercondition. After the hydrogel matrices are patterned with UV light, theCHO cell culture media can be loaded into the microfluidic channel. Thehydrogel matrix device can then be incubated at 37° C. FIG. 32 showsimages of the CHO cell culture at 0 hours, 18 hours, 42 hours, and 46hours. Fresh cell culture media was reloaded every 24 hours.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method of analyzing one or more biologicalcomponents of a biological sample, the method comprising: providing afluidic device comprising (i) a channel comprising a first surface, abiological sample, and one or more polymer precursors, wherein one ormore biological components of the biological sample are disposed on oradjacent to the first surface, (ii) a spatial energy modulating elementin optical communication with the first surface, and (iii) a detectorthat identifies positions of the one or more biological components inthe channel based on one or more optical signals therefrom; andsynthesizing one or more chambers in the channel enclosing each of theone or more biological components by projecting light into the channelwith the spatial energy modulating element such that the projected lightcauses cross-linking of the one or more polymer precursors to formpolymer matrix walls of the chambers, wherein the position of each ofthe synthesized chambers is determined by the position of a biologicalcomponent enclosed thereby identified by the detector.
 2. The method ofclaim 1 wherein said biological sample comprises one or more cells andwherein said first surface comprises one or more capture elements forcapturing one or more biological components of said biological sample.3. The method of claim 2 wherein said one or more cells comprise one ormore mammalian cells.
 4. The method of claim 2 further comprisingloading into said channel a lysing reagent so that said one or morecells in said chambers are lysed and said biological components of saidone or more cells is released and captured by said capture elements. 5.The method of claim 4 wherein said biological components compriseintracellular proteins.
 6. The method of claim 4 wherein said biologicalcomponents comprise RNA or DNA and wherein said capture elementscomprise capture oligonucleotides that capture such RNA or DNA.
 7. Themethod of claim 6 wherein said RNA comprises messenger RNA.
 8. Themethod of claim 7 further comprising depolymerizing said polymer matrixwalls of said chambers and loading said channel with reversetranscriptase reagents to copy said captured messenger RNAs to producecomplementary DNAs.
 9. The method of claim 8 further comprising: (a)amplifying said complementary DNAs, (b) sequencing the amplifiedcomplementary DNAs, and (c) determining a transcriptome of each of saidcells of each of said chambers.
 10. The method of claim 9 wherein saidamplifying comprises bridge amplifying each of said complementary DNAsto form a cluster for each thereof and wherein said sequencing comprisessequencing by synthesis said complementary DNA of each cluster.
 11. Themethod of claim 4 wherein (i) said capture elements comprise barcodeseach of which indicate a position thereof on said first surface, (ii)said biological components comprise RNA or DNA, and (iii) said captureelements comprise capture oligonucleotides that capture such RNA or DNA.12. The method of claim 11 wherein said RNA comprises messenger RNA andfurther comprising depolymerizing said polymer matrix walls of saidchambers and loading said channel with reverse transcriptase reagents tocopy the captured messenger RNAs to produce complementary DNAs.
 13. Themethod of claim 12 further comprising: (a) sequencing the complementaryDNAs, and (b) determining a transcriptome of each of said cells of eachof said chambers from the sequences of the complementary DNAs.
 14. Themethod of claim 13 wherein each of said barcodes further comprises auniversal molecular identifier.
 15. The method of claim 2 wherein saidcapture elements comprise barcodes indicating a position thereof on saidfirst surface and further comprising (i) incubating said one or morecells with antibodies specific for surface proteins whose relativeexpression permits identification of said cells, wherein each of suchantibodies has an oligonucleotide label comprising an antibody-specificbarcode capable of being captured by said capture element; and (ii)after said synthesizing, loading into said channel a lysing reagent sothat said one or more cells in said chambers are lysed, so thatoligonucleotide labels of antibodies attached to said one or more cellsare released and captured by said capture elements.
 16. The method ofclaim 15 further comprising depolymerizing said polymer matrix walls ofsaid chambers and loading said channel with reagents to copy saidcaptured oligonucleotide labels and said barcode of said capture elementto produce complementary DNAs each with said barcode indicating positionon said first surface.
 17. The method of claim 16 further comprising:(a) amplifying said complementary DNAs and barcodes (b) sequencing theamplified complementary DNAs and barcodes, and (c) determining relativeexpression of said surface proteins for each of said cells of each ofsaid chambers.
 18. The method of claim 17 wherein said antibodies areselected to identify said one or more cells as CD8+ T cells, monocytes,CD4+ T cells, NK cells, DC cells and B cells.
 19. The method of claim 1(i) wherein said biological sample comprises one or more cells secretingproteins, (ii) further comprising combining therewith protein-capturebeads comprising protein-capture antibodies; (iii) wherein saidsynthesizing further includes enclosing each of the one or more cellswith one or more protein-capture beads adjacent thereto; and (iv)further comprising, after said synthesizing, (A) incubating the one ormore cells secreting proteins with the protein-capture beads so thatsecreted proteins are captured by adjacent protein-capture beads; and(B) loading labeled protein detection antibodies and detecting proteinsecreted by each of the one or more cells by an amount of labeledprotein detection antibodies on protein-capture beads adjacent to eachof the one or more cells.
 20. The method of claim 1 wherein said polymermatrix walls are degradable by treating with a degradation agent andwherein said method further comprises: identifying one or more of saidchambers comprising said biological components that have selectedcharacteristics based on said one or more optical signals; and degradingsaid polymer matrix walls of the identified one or more said chambersbased on the selected characteristics.
 21. The method of claim 20wherein said polymer matrix walls comprise photocleavable crosslinkerscleavable by exposure to light of a different wavelength than that ofsaid wavelength of light used to synthesize said polymer matrix walls.22. The method of claim 20 wherein said polymer matrix walls compriseacid labile crosslinkers and a photoacid generators and wherein saiddegrading comprises selectively directing a beam of light to saididentified one or more chambers to stimulate the photoacid generatorstherein to generate acid to cleave the acid labile crosslinkers.
 23. Themethod of claim 20 wherein said polymer matrix walls comprise baselabile crosslinkers and photobase generators and wherein said degradingcomprises selectively directing a beam of light to said identified oneor more chambers to stimulate the photobase generators therein togenerate basic conditions to cleave the base labile crosslinkers. 24.The method of claim 1 wherein said polymer matrix walls are degradableby treating with a degradation agent and wherein said method furthercomprises: identifying one or more of said chambers comprising saidbiological components that have selected characteristics based on saidone or more optical signals; loading said channel with a second reactionmixture comprising second polymer precursors, wherein the second polymerprecursors are capable of forming second polymer matrix walls which arenondegradable for at least one degradation agent; and synthesizingsecond chambers enclosing the identified chambers.
 25. The method ofclaim 24 further comprising degrading said chambers, thereby separatingsaid biological components comprising said selected characteristics. 26.The method of claim 1 wherein said channel comprises an opticallytransmissive material.
 27. The method of claim 1 wherein said spatialenergy modulating element is a digital micromirror device.
 28. Themethod of claim 1 wherein said biological components are disposedrandomly on said first surface.
 29. The method of claim 1 wherein saidchannel of said fluidic device further comprises a second surfacewherein said first surface and the second surface are disposed oppositeone another across said channel, and wherein said polymer matrix wallsof said chambers extend from said first surface to the second surface toform one or more chambers each comprising an interior.
 30. The method ofclaim 29 wherein said interiors of a portion of said one or morechambers each contains a biological component.